Detoxified pneumolysin derivative ΔA146Ply inhibits autophagy and induces apoptosis in acute myeloid leukemia cells by activating mTOR signaling

Leukemia is caused by the malignant clonal expansion of hematopoietic stem cells, and in adults, the most common type of leukemia is acute myeloid leukemia (AML). Autophagy inhibitors are often used in preclinical and clinical models in leukemia therapy. However, clinically available autophagy inhibitors and their efficacy are very limited. More effective and safer autophagy inhibitors are urgently needed for leukemia therapy. In a previous study, we showed that ΔA146Ply, a mutant of pneumolysin that lacks hemolytic activity, inhibited autophagy of triple-negative breast cancer cells by activating mannose receptor (MR) and toll-like receptor 4 (TLR4) and that tumor-bearing mice tolerated ΔA146Ply well. Whether this agent affects AML cells expressing TLR4 and MR and the related mechanisms remain to be determined. In this study, we found that ΔA146Ply inhibited autophagy and induced apoptosis in AML cells. A mechanistic study showed that ΔA146Ply inhibited autophagy by activating mammalian target of rapamycin signaling and induced apoptosis by inhibiting autophagy. ΔA146Ply also inhibited autophagy and induced apoptosis in a mouse model of AML. Furthermore, the combination of ΔA146Ply and chloroquine synergistically inhibited autophagy and induced apoptosis in vitro and in vivo. Overall, this study provides an alternative effective autophagy inhibitor that may be used for leukemia therapy.Subject terms: Translational research, Acute myeloid leukaemia     

Structure-Based Virtual Screening towards the Discovery of Novel ULK1 Inhibitors with Anti-HCC Activities

There is an urgent need to develop new effective therapies for HCC. Our previous study identified ULK1 as the potential target for HCC therapy and screened the compound XST-14 as a specific inhibitor of ULK1 to suppress HCC progression. However, the poor manufacturability of XST-14 impeded the process of its clinical translation. In this study, we first generated pharmacophore models of ULK1 based on the X-ray structure of UKL1 in complex with ligands. We then screened the Specs chemical library for potential UKL1 inhibitors. By molecular docking, we screened out the 19 compounds through structure-based virtual screening. Through CCK8 activity screening on HCC cells, we found that ZZY-19 displayed obvious cell killing effects on HCC cells. SPR assay indicated that ZZY-19 had a higher binding affinity for ULK1 than XST-14. Moreover, ZZY-19 induced the effects of anti-proliferation, anti-invasion and anti-migration in HCC cells. Mechanistically, ZZY-19 induces autophagy inhibition by reducing the expression of ULK1 on HCC cells. Especially, the combination of ZZY-19 with sorafenib synergistically suppresses the progression of HCC in vivo. Taken together, ZZY-19 was a potential candidate compound that targeted ULK1 and possessed promising anti-HCC activities by inhibiting autophagy.     

Discovery of Monoacylglycerol Lipase (MAGL) Inhibitors Based on a Pharmacophore-Guided Virtual Screening Study

Monoacylglycerol lipase (MAGL) is an important enzyme of the endocannabinoid system that catalyzes the degradation of the major endocannabinoid 2-arachidonoylglycerol (2-AG). MAGL is associated with pathological conditions such as pain, inflammation and neurodegenerative diseases like Parkinson’s and Alzheimer’s disease. Furthermore, elevated levels of MAGL have been found in aggressive breast, ovarian and melanoma cancer cells. Due to its different potential therapeutic implications, MAGL is considered as a promising target for drug design and the discovery of novel small-molecule MAGL inhibitors is of great interest in the medicinal chemistry field. In this context, we developed a pharmacophore-based virtual screening protocol combined with molecular docking and molecular dynamics simulations, which showed a final hit rate of 50% validating the reliability of the in silico workflow and led to the identification of two promising and structurally different reversible MAGL inhibitors, VS1 and VS2. These ligands represent a valuable starting point for structure-based hit-optimization studies aimed at identifying new potent MAGL inhibitors.     

A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery

We sought to develop a sensitive and quantitative technique capable of monitoring the entire flux of autophagy involving fusion of lysosomal membranes. We observed the accumulation inside lysosomal compartments of Keima, a coral-derived acid-stable fluorescent protein that emits different-colored signals at acidic and neutral pHs. The cumulative fluorescent readout can be used to quantify autophagy at a single time point. Remarkably, the technique led us to characterize an autophagy pathway in Atg5-deficient cells, in which conventional LC3-based autophagosome probes are ineffective. Due to the large Stokes shift of Keima, this autophagy probe can be visualized in conjunction with other green-emitting fluorophores. We examined mitophagy as a selective autophagic process; time-lapse imaging of mitochondria-targeted Keima and GFP-Parkin allowed us to observe simultaneously Parkin recruitment to and autophagic degradation of mitochondria after membrane depolarization.     

Molecules and their functions in autophagy

Autophagy is a self-degradation system of cellular components through an autophagosomal-lysosomal pathway. Over the last 15 yr, yeast genetic screens led to the identification of a number of genes involved in the autophagic pathway. Most of these autophagy genes are present in higher eukaryotes and regulate autophagy process for cell survival and homeostasis. Significant progress has recently been made to better understand the molecular mechanisms of the autophagy machinery. Especially, autophagy process, including the regulation of autophagy induction through mTOR and the nucleation and elongation in autophagosome formation through class III phosphatidylinositol 3-kinase complex and ubiquitin-like conjugation systems, became evident. While many unanswered questions remain to be answered, here, we summarize the recent process of autophagy with emphasis on molecules and their protein complexes along with advanced molecular mechanisms that regulate the autophagy machinery.     

Design,Synthesis and Pharmacological Evaluation of Novel Hsp90N-terminal Inhibitors Without Induction of Heat Shock Response

Heat shock protein 90 (Hsp90) is a potential oncogenic target. However, Hsp90 inhibitors in clinical trial induce heat shock response, resulting in drug resistance and inefficiency. In this study, we designed and synthesized a series of novel triazine derivatives ( A1 - 26 , B1 - 13 , C1 - 23 ) as Hsp90 inhibitors. Compound A14 directly bound to Hsp90 in a different manner from traditional Hsp90 inhibitors, and degraded client proteins, but did not induce the concomitant activation of Hsp72. Importantly, A14 exhibited the most potent anti-proliferation ability by inducing autophagy, with the IC₅₀ values of 0.1 μM and 0.4 μM in A549 and SK-BR-3 cell lines, respectively. The in vivo study demonstrated that A14 could induce autophagy and degrade Hsp90 client proteins in tumor tissues, and exhibit anti-tumor activity in A549 lung cancer xenografts. Therefore, the compound A14 with potent antitumor activity and unique pharmacological characteristics is a novel Hsp90 inhibitor for developing anticancer agent without heat shock response.     

Evaluation of 3- and 4-Phenoxybenzamides as Selective Inhibitors of the Mono-ADP-Ribosyltransferase PARP10

Intracellular ADP-ribosyltransferases catalyze mono- and poly-ADP-ribosylation and affect a broad range of biological processes. The mono-ADP-ribosyltransferase PARP10 is involved in signaling and DNA repair. Previous studies identified OUL35 as a selective, cell permeable inhibitor of PARP10. We have further explored the chemical space of OUL35 by synthesizing and investigating structurally related analogs. Key synthetic steps were metal-catalyzed cross-couplings and functional group modifications. We identified 4-(4-cyanophenoxy)benzamide and 3-(4-carbamoylphenoxy)benzamide as PARP10 inhibitors with distinct selectivities. Both compounds were cell permeable and interfered with PARP10 toxicity. Moreover, both revealed some inhibition of PARP2 but not PARP1, unlike clinically used PARP inhibitors, which typically inhibit both enzymes. Using crystallography and molecular modeling the binding of the compounds to different ADP-ribosyltransferases was explored regarding selectivity. Together, these studies define additional compounds that interfere with PARP10 function and thus expand our repertoire of inhibitors to further optimize selectivity and potency.     

A Quassinoid Diterpenoid Eurycomanone from Eurycoma longifolia Jack Exerts Anti-Cancer Effect through Autophagy Inhibition

Eurycomanone (EN) is one of the representative quassinoid diterpenoids from roots of Eurycoma longifolia Jack, a natural medicine that is widely distributed in Southeast Asia. Previous studies showed that EN induces cancer cell apoptosis and exhibits anti-cancer activity, but the molecular mechanism of EN against cancer has still not been elucidated. In this study, we examined the regulatory effect of EN on autophagy to reveal the mechanism of EN-mediated colon cancer growth inhibition. First, we found that EN is able to inhibit colon cancer cell proliferation and colony formation. The angiogenesis level in cancer cells was inhibited as well. Next, the treatment of EN led to the suppression of autophagy, which was characterized by the downregulation of the LC3-II level and the formation of GFP-LC3 puncta under EN treatment in colon cancer. Moreover, we revealed that the mTOR signaling pathway was activated by EN in a time- and concentration-dependent manner. Finally, autophagy induction protected colon cancer cells from EN treatment, suggesting that autophagy improves cell survival. Taken together, our findings revealed the mechanism of EN against colon cancer through inhibiting autophagy and angiogenesis in colon cancer, supporting that the autophagy inhibitor EN could be developed to be a novel anti-cancer agent.     

Docking-based virtual screening studies aiming at the covalent inhibition of SARS-CoV-2 MPro by targeting the cysteine 145

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 which has infected millions of people worldwide. The main protease of SARS-CoV-2 (M^Pro) has been recognized as a key target for the development of antiviral compounds. Taking advantage of the X-ray crystal complex with reversible covalent inhibitors interacting with the catalytic cysteine 145 (Cys145), we explored flexible docking studies to select alternative compounds able to target this residue as covalent inhibitors. First, docking studies of three known electrophilic compounds led to results consistent with co-crystallized data validating the method for SARS-CoV-2 M^Pro covalent inhibition. Then, libraries of soft electrophiles (overall 41 757 compounds) were submitted to docking-based virtual screening resulting in the identification of 17 molecules having their electrophilic group close to the Cys145 residue. We also investigated flexible docking studies of a focused approved covalent drugs library including 32 compounds with various electrophilic functional groups. Among them, the calculations resulted in the identification of four compounds, namely dimethylfumarate, fosfomycin, ibrutinib and saxagliptin, able first, to bind to the active site of the protein and second, to form a covalent bond with the catalytic cysteine.     

Identification of potential inhibitors against nuclear Dam1 complex subunit Ask1 of Candida albicans using virtual screening and MD simulations

Identification of hit compounds against specific target form the starting point for a drug discovery program. A consistent decline of new chemical entities (NCEs) in recent years prompted a challenge to explore newer approaches to discover potential hit compounds that in turn can be converted into leads, and ultimately drug with desired therapeutic efficacy. The vast amount of omics and activity data available in public databases offers an opportunity to identify novel targets and their potential inhibitors. State of the art in silico methods viz., clustering of compounds, virtual screening, molecular docking, MD simulations and MMPBSA calculations were employed in a pipeline to identify potential ‘hits’ against those targets as well whose structures, as of now, could only predict through threading approaches. In the present work, we have started from scratch, amino acid sequence of target and compounds retrieved from PubChem compound database, modeled it in such a way that led to the identification of possible inhibitors of Dam1 complex subunit Ask1 of Candida albicans. We also propose a ligand based binding site determination approach. We have identified potential inhibitors of Ask1 subunit of a Dam1 complex of C. albicans, which is required to prevent precocious spindle elongation in pre-mitotic phases. The proposed scheme may aid to find virtually potential inhibitors of other unique targets against candida.     

Discovery of Novel Cinchona‐Alkaloid‐Inspired Oxazatwistane Autophagy Inhibitors

The cinchona alkaloids are a privileged class of natural products and are endowed with diverse bioactivities. However, for compounds with the closely‐related oxazatricyclo[4.4.0.0]decane (“oxazatwistane”) scaffold, which are accessible from cinchonidine and quinidine by means of ring distortion and modification, biological activity has not been identified. We report the synthesis of an oxazatwistane compound collection through employing state‐of‐the‐art C−H functionalization, and metal‐catalyzed cross‐coupling reactions as key late diversity‐generating steps. Exploration of oxazatwistane bioactivity in phenotypic assays monitoring different cellular processes revealed a novel class of autophagy inhibitors termed oxautins, which, in contrast to the guiding natural products, selectively inhibit autophagy by inhibiting both autophagosome biogenesis and autophagosome maturation.     

A time-resolved fluorescence probe for dipeptidyl peptidase 4 and its application in inhibitor screening

The prevalence of type 2 diabetes is increasing dramatically throughout the world. Recently, dipeptidyl peptidase 4 (DPP4) was identified as a potential antidiabetes target. Many DPP4 inhibitors, such as sitagliptin and vildagliptin, have been developed and marketed, but superior therapeutic agents are still required. Therefore, we have developed new methodology for screening of DPP4 inhibitors. Absorption-based measurements with para-nitroaniline or fluorescence-based measurements with the coumarin derivative 7-amino-4-methylcoumarin are often used for the screening of protease inhibitors, including DPP4 inhibitors, but these strategies are not sufficiently sensitive because of interfering background absorption and fluorescence, thus giving rise to many false-positive and false-negative results. Therefore, we have designed and synthesised a novel DPP4 probe (Gly-Pro-BCD-Tb; Gly=glycine, Pro=proline, andBCD defines the backbone of the probe comprising an aniline derivative as on/off switch, a 7-amino-4-methyl-2(1H)-quinolinone (cs-124) as antenna moiety, and a diethylenetriamine-N,N,N',N',N'-pentaacetic acid (DTPA) as chelator moiety, Tb=terbium) for time-resolved fluorescence (TRF) measurements. TRF measurements with Gly-Pro-BCD-Tb showed high sensitivity and reliability in the inhibitory assay relative to Gly-Pro-MCA (MCA=4-methylcoumarin-7-amide), a conventional fluorescence probe for DPP4. Further, we employed our probe for high-throughput DPP4 inhibitor screening with 3841 randomly selected compounds and found that epibestatin, an epimer of bestatin (a well-known anticancer drug and general aminopeptidase inhibitor), showed dose-dependent DPP4 inhibitory activity. Interestingly, bestatin did not exhibit DPP4 inhibitory activity. We believe that this screening system will be useful for the discovery of DPP4 inhibitors with novel structural scaffolds.     

The death of human cancer cells following photodynamic therapy: apoptosis competence is necessary for Bcl-2 protection but not for induction of autophagy

Photodynamic therapy (PDT) is an efficient inducer of apoptosis in many types of cells, except in cells deficient in one or more of the factors that mediate apoptosis. Recent reports have identified autophagy as a potential alternative cell death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro-apoptotic factor Bax (human prostate cancer DU145 cells) or the apoptosis mediator caspase-3 (human breast cancer MCF-7v cells) and in apoptosis-competent cells (MCF-7c3 cells that stably overexpress human pro-caspase-3 and Chinese hamster ovary CHO 5A100 cells). Further, each of the cell lines was also studied with and without stably overexpressed Bcl-2. Autophagy was identified by electron microscopic observation of the presence of double-membrane-delineated autophagosomal vesicles in the cytosol and by immunoblot observation of the Pc 4-PDT dose- and time-dependent increase in the level of LC3-II, a component of the autophagosomal membrane. Autophagy was observed in all of the cell lines studied, whether or not they were capable of typical apoptosis and whether or not they overexpressed Bcl-2. The presence of stably overexpressed Bcl-2 in the cells protected against PDT-induced apoptosis and loss of clonogenicity in apoptosis-competent cells (MCF-7c3 and CHO 5A100 cells). In contrast, Bcl-2 overexpression did not protect against the development of autophagy in any of the cell lines or against loss of clonogenicity in apoptosis-deficient cells (MCF-7v and DU145 cells). Furthermore, 3-methyladenine and wortmannin, inhibitors of autophagy, provided greater protection against loss of viability to apoptosis-deficient than to apoptosis-competent cells. The results show that autophagy occurs during cell death following PDT in human cancer cells competent or not for normal apoptosis. Only the apoptosis-competent cells are protected by Bcl-2 against cell death.     

Screening and docking studies of natural phenolic inhibitors of carbonic anhydrase II

Carbonic anhydrase II (CA II) is an important enzyme complex with Zn²⁺, which is involved in many physiological and pathological processes, such as calcification, glaucoma and tumorigenicity. In order to search for novel inhibitors of CA II, inhibition assay of carbonic anhydrase II was performed, by which seven natural phenolic compounds, including four phenolics (grifolin, 4-O-methyl-grifolic acid, grifolic acid, and isovanillic acid) and three flavones (eriodictyol, quercetin and puerin A), showed inhibitory activities against CA II with IC₅₀s in the range of 6.37–71.73 μmol/L. Grifolic acid is the most active one with IC₅₀ of 6.37 _μmol/L. These seven phenolic compounds were proved to be novel natural carbonic anhydrase II inhibitors, which were obtained in flexible docking study with GOLD 3.0 software. Results indicated that the aliphatic chain and polar groups of hydroxyl and carboxyl are important to their inhibitory activities, providing a new insight into study on CA II potent inhibitors. Authors with the equal contribution Supported by the National Natural Science Foundation of China (Grant No. 30725048) and the Foundation of Chinese Academy of Sciences (West Light Program).     

Triflumizole as a Novel Lead Compound for Strigolactone Biosynthesis Inhibitor

Strigolactones (SLs) are carotenoid-derived plant hormones involved in the development of various plants. SLs also stimulate seed germination of the root parasitic plants, Striga spp. and Orobanche spp., which reduce crop yield. Therefore, regulating SL biosynthesis may lessen the damage of root parasitic plants. Biosynthetic inhibitors effectively control biological processes by targeted regulation of biologically active compounds. In addition, biosynthetic inhibitors regulate endogenous levels in developmental stage- and tissue-specific manners. To date, although some chemicals have been found as SL biosynthesis inhibitor, these are derived from only three lead chemicals. In this study, to find a novel lead chemical for SL biosynthesis inhibitor, 27 nitrogen-containing heterocyclic derivatives were screened for inhibition of SL biosynthesis. Triflumizole most effectively reduced the levels of rice SL, 4-deoxyorobanchol (4DO), in root exudates. In addition, triflumizole inhibited endogenous 4DO biosynthesis in rice roots by inhibiting the enzymatic activity of Os900, a rice enzyme that converts the SL intermediate carlactone to 4DO. A Striga germination assay revealed that triflumizole-treated rice displayed a reduced level of germination stimulation for Striga. These results identify triflumizole as a novel lead compound for inhibition of SL biosynthesis.     

Carborane-Based Analog of Rev-5901 Attenuates Growth of Colon Carcinoma In Vivo

Lipoxygenases convert polyunsaturated fatty acids into biologically active metabolites such as inflammatory mediators—prostaglandins and leukotrienes. The inhibition of lipoxygenases is increasingly employed in the treatment of cancer. We evaluated the anticancer potential of two novel 5-lipoxygenase inhibitors, named CarbZDNaph and CarbZDChin, which are analogues of the commercially available inhibitor Rev-5901. The in vitro segment of this study was conducted on a mouse colorectal carcinoma cell line—CT26CL25. For an in vivo model, we induced tumors in BALB/c mice by the implantation of CT26CL25 cells, and we treated the animals with potential inhibitors. A 48 h treatment resulted in diminished cell viability. Calculated IC₅₀ values (half-maximal inhibitory concentrations) were 25 μM, 15 μM and 30 μM for CarbZDNaph, CarbZDChin and Rev-5901, respectively. The detailed analysis of mechanism revealed an induction of caspase-dependent apoptosis and autophagy. In the presence of chloroquine, an autophagy inhibitor, we observed an increased mortality of cells, implying a cytoprotective role of autophagy. Our in vivo experiment reports tumor growth attenuation in animals treated with CarbZDChin. Compounds CarbZDNaph and Rev-5901 lacked an in vivo efficacy. The results presented in this study display a strong effect of compound CarbZDChin on malignant cell growth. Having in mind the important role of inflammation in cancer development, these results have a significant impact and are worthy of further evaluation.     

Selective ATP competitive leads of CDK4: Discovery by 3D-QSAR pharmacophore mapping and molecular docking approach

The discovery of ATP competitive CDK4 inhibitors is an on-going challenging task in cancer therapy. Here, an attempt has been made to develop new leads targeting ATP binding site of CDK4 by applying 3D-QSAR pharmacophore mapping and molecular docking methods The outcome of 6 leads offers a significant contribution for selective CDK4 inhibition, since they show potential binding interactions with Val⁹⁶, Arg¹⁰¹, and Glu¹⁴⁴ residues of CDK4, that are unique and from other kinases. It is worth noting that there is a striking similarity in binding interactions of the leads and known CDK4 inhibitors, namely Abemaciclib, Palbociclib and Ribociclib. Further key features, including high dock score value, good predicted activity, scaffold diversity, and the acceptable ADME profile of leads, provide a great opportunity for the development of highly potent and selective ATP competitive inhibitors of CDK4.     

Synthesis and structure-activity relationship of N-arylrolipram derivatives as inhibitors of PDE4 isozymes

Structure activity studies of N-phenylrolipram derivatives have led to the identification of highly potent PDE4 inhibitors. The potential of these inhibitors for cellular activity was routinely assessed in an assay of fMLP induced oxidative burst in human eosinophils. Since first generation PDE4 inhibitors have been plagued with a number of unwanted side effects, parallel structure activity studies for competition with the [3H]-rolipram binding site in rat brain were performed. In this fashion 5-[4-(3-cyclopentyloxy-4-methoxyphenyl)-2-oxo-pyrrolidin-1-yl]-3-(3-methoxybenzyloxy)benzoic acid N',N'-dimethylhydrazide (22) was identified as a potent inhibitor of PDE4 which exhibits >1000 fold selectivity versus PDE3, and is a nanomolar inhibitor in all the cellular assays tested. Studies on the stereoselectivity of PDE4 inhibition of this class of rolipram based compounds revealed, that for example (S)-11 is a more potent inhibitor than (R)-11. This effect can also be observed in primary human cells where the (S)-enantiomer is about 10 fold more potent than the corresponding (R)-enantiomer.