Synthesis of 2-phenylnaphthalenoid amide derivatives and their topoisomerase IIα inhibitory and antiproliferative activities.

Topoisomerases are highly associated with cell proliferation, becoming an important target for the development of antitumor drugs. 2-Phenylnaphthalenoids (2PNs) have been identified as human DNA topoisomerase IIα (TopoIIα) inhibitors. In this study, based on the 2PN scaffold, 20 amide derivatives (J1-J10, K1-K10) were synthesized. Among them, K10 showed high TopoIIα inhibitory activity and stronger antiproliferation activity against HepG-2 and MDA-MB-231 cells (IC50 0.33 and 0.63 µM, respectively) than the positive control VP-16 (IC50 9.19 and 10.86 µM) and the lead F2 (IC50 0.64 and 1.51 µM). Meanwhile, K10 could also inhibit migration and promote apoptosis of HepG-2 and MDA-MB-231 cells. Therefore, K10 can be developed into a potent TopoIIα inhibitor as an antitumor agent. The structure-activity relationship was also discussed.

Proansamycin B derivatives from the post-PKS modification gene deletion mutant of Amycolatopsis mediterranei S699.

Ten new proansamycin B congeners (1-10) together with one known (11) were isolated and characterized on the basis of 1D and 2D NMR spectroscopic and HRESIMS data from the Amycolatopsis mediterranei S699 ΔPM::rifR+rif-orf19 mutant. Compounds 8 and 9 featured with six-membered ring and five-membered ring hemiketal, respectively. Compounds 1, 2, and 9 displayed antibacterial activity against MRSA (methicillin-resistant Staphylococcus aureus), with the MIC (minimal inhibitory concentration) values of 64, 8, and 128 µg/mL, respectively. Compound 1 showed significant cytotoxicity against MDA-MB-231, HepG2 and Panc-1 cell lines with IC50 (half maximal inhibitory concentration) values of 2.3 ± 0.2, 2.5 ± 0.3 and 3.8 ± 0.5 µM, respectively.

Phosphatase, Mg2+/Mn2+ dependent 1B regulates the hematopoietic stem cells homeostasis via the Wnt/ß-catenin signaling.

Hematopoietic stem cells (HSCs) are primarily dormant in a cell-cycle quiescence state to preserve their self-renewal capacity and long-term maintenance. How HSCs maintain the balance between activation and quiescence remains largely unknown. Herein, we found that Phosphatase, Mg2+/Mn2+ Dependent 1B (Ppm1b) is required for the expansion of phenotypic HSCs in vitro. By using a conditional knockout mouse model in which Ppm1b was specifically depleted in hematopoietic cells, we demonstrated that loss of Ppm1b impaired the HSC homeostasis and hematopoietic reconstitution. Ppm1b deficiency mice also exhibited B-cell leukocytopenia, which is due to the compromised commitment and proliferation of B-biased lymphoid progenitor cells from CLPs. With the aid of a small molecular inhibitor, we confirmed the roles of Ppm1b in adult hematopoiesis that phenocopied the effects with loss of Ppm1b. Furthermore, transcriptome profiling of Ppm1b-deficient HSCs revealed the disruptive quiescence of HSC. Mechanistically, Ppm1b interacted with ß-catenin and mediated its dephosphorylation. Loss of Ppm1b led to the decrease of the active ß- catenin (non-phosphorylated) that interrupted the Wnt/ß-catenin signaling in HSC, which consequently suppressed HSC expansion. Together, our study identified an indispensable role of Ppm1b in regulating HSC homeostasis via Wnt/ß-catenin pathway.

Synthesis of urolithin derivatives and their anti-inflammatory activity.

Two series of urolithin derivatives, totally 38 compounds, were synthesized. Their anti-inflammatory activity was investigated by detecting the inhibitory effects on the expression of TNF-α in bone marrow-derived macrophages (BMDMs), showing that 24 of 38 ones reduced the expression of TNF-α. Compound B2, the ring C opened derivative of urolithin B with a butoxycarbonyl substitution in ring A, showed the strongest inhibitory activity compared with that of indomethacin. Furthermore, B2 treatment decreased the expression of pro-inflammatory factors IL-1ß, IL-6, iNOS and COX-2. Mechanically, the anti-inflammatory effect of B2 was related to the inhibition of NF-κB signaling pathway. These results clearly illustrated that B2 hold potential for application as an anti-inflammatory agent. The present study provided a viable approach to modify the gut metabolites for anti-inflammatory drug development.

Resveratrol-derived inhibitors of the E3 ubiquitin ligase PELI1 inhibit the metastasis of triple-negative breast cancer.

Triple-negative breast cancer (TNBC), as the most challenging subtype of breast cancer, exerts highly invasive ability and metastatic nature to the lymph nodes, which is correlated with poor survival rates among patients. Pellino-1 (PELI1) is an E3 ubiquitin ligase involved in tumor invasion and metastasis, and has the potential to be developed as a novel therapeutic target for TNBC. In this study, we identified a potent inhibitor of PELI1, namely compound 3d, on the basis of natural stilbene framework through medicinal chemistry approaches. This novel PELI1 inhibitor 3d showed potent binding affinity to PELI1 (Kd 8.2 µM) in fluorescence quenching assay, and markedly interrupted the interaction of PELI1 and SNAIL/SLUG confirmed by co-immunoprecipitation. Moreover, 3d exhibited potent antitumor activity in inhibiting tumor cell migration in scratch wound healing assay without affecting cell proliferation in vitro, and down-regulated the downstream EMT-effectors of PELI1 as assessed by western blotting. In the experimental lung metastasis model, 3d showed anti-TNBC metastasis efficacy without observable toxicity in vivo.

PELI2 regulates early B-cell progenitor differentiation and related leukemia via the IL-7R expression.

Little is known about the transition mechanisms that govern early lymphoid lineage progenitors from common lymphoid progenitors (CLPs). Pellino2 (PELI2) is a newly discovered E3 ubiquitin ligase, which plays important roles in inflammation and immune system. However, the physiological and molecular roles of PELI2 in the differentiation of immune cells are largely unknown. Here, by using a conditional knockout mouse model, we demonstrated that PELI2 is required for the early B-cell development and stressed hematopoiesis. PELI2 interacted with and stabilized PU.1 via K63- polyubiquitination to regulate IL-7R expression. The defects of B cell development induced by PELI2 deletion were restored by overexpression of PU.1. Similarly, PELI2 promoted TCF3 protein stability via K63- polyubiquitination to regulate IL-7R expression, which is required for the proliferation of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells. These results underscore the significance of PELI2 in both normal B lymphopoiesis and malignant B-cell acute lymphoblastic leukemia via the regulation of IL-7R expression, providing a potential therapeutic approach for BCP-ALL.

Lapachol inhibits the growth of lung cancer by reversing M2-like macrophage polarization via activating NF-κB signaling pathway.

Resetting tumor-associated macrophages (TAMs) is a promising strategy to ameliorate the immunosuppressive tumor microenvironment (TME) and improve innate and adaptive antitumor immunity. Lapachol, a naturally occurring 1,4-naphthoquinone, exhibits various pharmacological activities including antitumor, anti-leishmanial, antimalarial and antiseptic. In this study, we investigated the relevance of macrophage polarization and the antitumor effect of lapachol in Lewis lung cancer (LLC) both in vitro and in vivo. This study demonstrated that lapachol significantly reversed the polarization of M2-like macrophages thus that were endowed with the ability to kill LLC cells by activating NF-κB signaling pathway. Furthermore, lapachol effectively suppressed tumor growth in C57BL/6 mice bearing lung tumors by reducing the proportion of M2-like macrophages. Overall, our findings clearly illustrated that lapachol could reverse the polarization of M2-like macrophages to improve the immunosuppressive tumor microenvironment, and had the potential to be developed as an immunomodulatory antitumor agent.

LZY3016, a novel geldanamycin derivative, inhibits tumor growth in an MDA-MB-231 xenograft model.

A novel geldanamycin derivative LZY3016 was synthesized as an antitumor agent. Compound LZY3016 exhibited potent anti-proliferation activity toward MDA-MB-231 (IC50 = 0.06 µM), which was more effective than positive drug 17-AAG. In vivo hepatotoxicity assay displayed that serum AST/ALT levels in LZY3016-treated mice were both significantly less than those in the geldanamycin (GA) group. LZY3016 showed potent antitumor activity in an MDA-MB-231 xenograft mouse model, suggesting LZY3016 is an up-and-coming antitumor candidate. The theoretical binding mode between LZY3016 and Hsp90 was obtained by molecular dynamics simulation.

PELI1 and EGFR cooperate to promote breast cancer metastasis.

Pellino-1 (PELI1) is an E3 ubiquitin ligase acting as a key regulator for the inflammation and autoimmunity via the ubiquitination of the substrate proteins. There is increasing evidence to support that PELI1 functions as an oncoprotein in tumorigenesis and metastasis. However, the molecular mechanism underlying the high expression and oncogenic roles of PELI1 in cancers remains limited. Herein, we revealed a novel regulation mechanism by which PELI1 and EGFR cooperate to promote breast cancer metastasis. EGFR is positively correlated with PELI1 expression in breast cancers, and its activation led to the phosphorylation of PELI1 at Tyr154 and Thr264, which subsequently activated its E3 ubiquitin ligase. Simultaneously, PELI1 physically interacted with and enhanced the stability of EGFR via the K63-linked polyubiquitination in reverse. The co-inhibition of the PELI1-EGFR showed synergetic effect to repress breast cancer metastasis. Furthermore, we identified a compound S62 as a small molecule disruptor of PELI1/EGFR that effectively repressed breast cancer metastasis. Our study not only uncovered the emerging roles of PELI1/EGFR interaction in the progression of breast cancer, but also provided an effective strategy for the inhibition of metastasis in breast cancer.

Identification and Characterization of the 28-N-Methyltransferase Involved in HSAF Analogue Biosynthesis.

Polycyclic tetramate macrolactams (PoTeMs) are a family of structurally intriguing bioactive natural products. Although the presence of the N-28 methyl group is known to affect bioactivities of some PoTeMs, the mechanism for this methylation remains unclear. We report here the identification and characterization of the 28-N-methyltransferase for HSAF analogues, which is encoded by a gene located outside the HSAF (heat-stable antifungal factor) cluster in Lysobacter enzymogenes C3. Our data suggested that 28-N-methyltransferase utilizes S-adenosylmethionine (SAM) to methylate HSAF analogues, and acts after the dicyclic and tricyclic ring formation and prior to C-3 hydroxylation. Kinetic analysis showed that the optimal substrate for the enzyme is 3-dehydroxy HSAF (3-deOH HSAF). Moreover, it could also accept PoTeMs bearing a 5-6 or 5-6-5 polycyclic system as substrates. This is the first N-methyltransferase identified in the family of PoTeMs, and the identification of this enzyme provides a new tool to generate new PoTeMs as antibiotic lead compounds.

Discovery of 1,2-diphenylethene derivatives as human DNA topoisomerase II catalytic inhibitors and antitumor agents.

Human DNA topoisomerase II (TopoII) is highly correlated with cell proliferation, and involved in tumor biogenesis and development. The classic chemotherapeutic agents etoposide (VP-16) and adriamycin (ADR) targeting TopoII are wildly used in clinical applications. Herein, fifty-eight pinosylvin (1,2-diphenylethene) derivatives as TopoII inhibitors were designed and synthesized through three generations of structural optimizations on the basis of the structure of the initial hit A1 from in-house chemical library. The most potent compound F2 showed high in vitro inhibitory efficacy against TopoII (IC50 α 3.8, ß 10.1 µM), compared to that of VP-16 (IC50 α 110.0, ß 36.1 µM) for pBR322 DNA relaxation with no evident TopoII poisons in DNA cleavage assay. Meanwhile, F2 exhibited strong antitumor activities against human cancer cell lines HeLa, HCT-116, PC-3, MDA-MB-231, HepG2 and A549 (IC50 0.1-0.3 µM), compared to that of VP-16 (IC50 1.5-15.1 µM). F2 showed less cytotoxicity against normal murine cell line CCL-226 (IC50 > 50 µM) than that of VP-16 (IC50 20.8 µM). The selectivity index of F2 and VP-16 are larger than 52.1 and 1.3-26.2 in cell lines, respectively. Additionally, F2 exhibited potent potency in apoptosis induction and cell cycle arrest in HepG2 cells. These results provide a promising strategy and good starting point for the development of potent TopoII inhibitors as antitumor agents.

Relationship between indices of circulating blood cells and bone homeostasis in osteoporosis.

Bone development have been shown to play an important role in regulating hematopoiesis as one major component of bone marrow microenvironment. Recent studies support the notion that there is an intricate relationship between hematopoiesis and bone homeostasis, however, little is known about the alterations in the hematopoietic lineages in pathologic conditions. Using various osteoporotic mouse models, we show here that bone microarchitecture abnormalities alter parameters of peripheral blood cells. The level of white blood cells is dynamics and negatively correlated with bone mineral density during the progression of osteoporosis. Furthermore, our clinical data confirm that osteoporosis is associated with abnormal circulating blood cell counts. These results demonstrated a causal link that osteoporosis is accompanied by the altered circulating blood cells, supporting the idea of a close interplay between hematopoiesis and bone homeostasis. Our study would propose that routine complete blood count might be applied as a potential diagnostic and putative marker for osteoporosis.

Combinatorial Biosynthesis of Oxidized Combamides Using Cytochrome P450 Enzymes from Different Polycyclic Tetramate Macrolactam Pathways.

Polycyclic tetramate macrolactams (PoTeMs) are a family of natural products containing a tetramic acid moiety and a polycyclic system. Due to the valuable biological activities of different PoTeMs and the genetic simplicity of their biosynthetic genes, it is highly desirable to manipulate the biosynthesis of PoTeMs by swapping modification genes between different pathways. Herein, by combining the cytochrome P450 (CYP) enzymes from different PoTeM pathways with the combamides' biosynthetic genes, the new combamides G (3), I (5), and J (6) along with the known combamides B (1), D (2), and H (4) were identified from the recombinant strains. Combamides G (3), H (4), and J (6) displayed cytotoxic activity against human cancer cell lines. Furthermore, our results demonstrated for the first time the substrate specificity of the PoTeM-related CYPs in vivo, which will facilitate the engineered biosynthesis of other PoTeMs in the future.

Targeted Discovery of the Polyene Macrolide Hexacosalactone A from Streptomyces by Reporter-Guided Selection of Fermentation Media.

New approaches are still needed to fully explore the biosynthetic potential of microbes. We recently devised a melC reporter-guided fermentation media screening approach for targeted activation of cryptic gene clusters. Using this approach, we successfully activated the expression of the hcl gene cluster in Streptomyces sp. LZ35 and discovered a novel polyene macrolide hexacosalactone A (1).

Triazole/thiadiazole substituted 4'-demethylepipodophyllotoxin derivatives induced apoptosis in HeLa cells by up-regulating TMEM133.

Heterocycle modification has been widely and successfully employed in the antitumor drugs. However, the different antitumor efficacy was corelated with the heterocycle substituted, and the genetic mechanism underlying these effects has not been elucidated. In this study, the intrinsic regularity between different types of heterocycle-substituted DMEP derivative compounds and the mechanisms of their antitumor activity was preliminarily disclosed. Triazole/thiadiazole substituted 4'-demethylepipodophyllotoxin derivatives induced more severe DNA damage and higher levels of 26S proteasomal Topo IIß degradation, though inhibited the recruition of γH2AX to resist the DNA damage. The reduced DNA repair led to higher up-regulation of cell cycle arrest proteins, and ultimately DNA damage mediated-ATM/ATR apoptotic pathways and specifically activated DNA damage response gene TMEM133, which induced apoptosis through up-regulation of G2/M cell cycle arrest-related genes. Over-expression and knock-out of TMEM133 demonstrated that TMEM133 is essential for inhibition of the tumor cell growth during treatment with triazole/thiadiazole substituted 4'-demethylepipodophyllotoxin derivatives.

The coupled reaction catalyzed by EchB and EchC lead to the formation of the common 2',3',5'-trihydroxy-benzene core in echosides biosynthesis.

p-Terphenyls represent a unique family of aromatic natural products generated by nonribosomal peptide synthetase-like (NRPS-like) enzyme. After formation of p-terphenyl skeleton, tailoring modifications will give rise to structural diversity and various biological activities. Here we demonstrated a two-enzyme (EchB, a short-chain dehydrogenase/reductase (SDR), and EchC, a nuclear transport factor 2 (NTF2)-like dehydratase) participated transformation from dihydroxybenzoquinone core to 2',3',5'-trihydroxy-benzene in the biosynthesis of echosides. Beginning with polyporic acid as substrate, successive steps of reduction-dehydration-reduction cascade catalyzed by EchB-EchC-EchB were concluded after in vivo gene disruption and in vitro bioassay experiments. These findings demonstrated a conserved synthesis pathway of 2',3',5'-trihydroxy-p-terphenyls in bacteria, such as Actinomycetes and Burkholderia. The parallel pathway in fungi has yet to be explored.

Challenges and potential for improving the druggability of podophyllotoxin-derived drugs in cancer chemotherapy.

Covering: up to 2020As a main bioactive component of the Chinese, Indian, and American Podophyllum species, the herbal medicine, podophyllotoxin (PTOX) exhibits broad spectrum pharmacological activity, such as superior antitumor activity and against multiple viruses. PTOX derivatives (PTOXs) could arrest the cell cycle, block the transitorily generated DNA/RNA breaks, and blunt the growth-stimulation by targeting topoisomerase II, tubulin, or insulin-like growth factor 1 receptor. Since 1983, etoposide (VP-16) is being used in frontline cancer therapy against various cancer types, such as small cell lung cancer and testicular cancer. Surprisingly, VP-16 (ClinicalTrials NTC04356690) was also redeveloped to treat the cytokine storm in coronavirus disease 2019 (COVID-19) in phase II in April 2020. The treatment aims at dampening the cytokine storm and is based on etoposide in the case of central nervous system. However, the initial version of PTOX was far from perfect. Almost all podophyllotoxin derivatives, including the FDA-approved drugs VP-16 and teniposide, were seriously limited in clinical therapy due to systemic toxicity, drug resistance, and low bioavailability. To meet this challenge, scientists have devoted continuous efforts to discover new candidate drugs and have developed drug strategies. This review focuses on the current clinical treatment of PTOXs and the prospective analysis for improving druggability in the rational design of new generation PTOX-derived drugs.

Identification of HN252 as a potent inhibitor of protein phosphatase PPM1B.

Protein phosphatase 1B (PPM1B), a member of metal-dependent protein serine/threonine phosphatase family, is involved in the regulation of several signalling pathways. However, our understanding of its substrate interaction and physiological functions is still largely limited. There is no reported PPM1B inhibitor to date. In this study, we identified HN252, a p-terphenyl derivative, as a potent PPM1B inhibitor (Ki  = 0.52 ± 0.06 µM). HN252 binding to PPM1B displayed remarkable and specific inhibition of PPM1B in both in vitro and ex vivo. With the aid of this small molecular inhibitor, we identified 30 proteins' serine/threonine phosphorylation as potential substrates of PPM1B, 5 of which were demonstrated by immunoprecipitation, including one known (CDK2) and 4 novel ones (AKT1, HSP90B, ß-catenin and BRCA1). Furthermore, GO and KEGG analysis of dramatically phosphorylated proteins by PPM1B inhibition indicated that PPM1B plays roles in the regulation of multiple cellular processes and signalling pathways, such as gene transcription, inflammatory regulation, ageing and tumorigenesis. Our work provides novel insights into further investigation of molecular mechanisms of PPM1B.

Expression of the Clifednamide Biosynthetic Pathway in Streptomyces Generates 27,28-seco-Derivatives.

Polycyclic tetramate macrolactams (PoTeMs) are a group of hybrid PK-NRP natural products having a variable set of carbocyclic rings, a conserved assembly pathway, and diverse bioactivities. We report here the identification of seven new PoTeMs, clifednamides D-J (3-9), along with the known clifednamides A (1) and B (2) through rational pathway refactoring and heterologous expression. Remarkably, clifednamides D (3), G (6), and H (7) feature an unprecedented 27,28-seco skeleton. The cytotoxic activities of compounds 1-9 indicated that the hydroxy group of C-25, the methyl group of C-30, the inner five-membered ring, and the intact macrocycle are all critical for the activities. Meanwhile, the cytochrome P450 enzyme CftS023A and the hydroxylase CftS023E involved in oxidative tailoring of clifednamides were found to decorate the fused 5-6 bicyclic intermediates. Accordingly, the biosynthetic pathway for clifednamides was proposed.

8-Deoxy-Rifamycin Derivatives from Amycolatopsis mediterranei S699 ΔrifT Strain.

Proansamycin X, a hypothetical earliest macrocyclic precursor in the biosynthesis of rifamycin, had never been isolated and identified. According to bioinformatics analysis, it was proposed that RifT (a putative NADH-dependent dehydrogenase) may be a candidate target responsible for the dehydrogenation of proansamycin X. In this study, the mutant strain Amycolatopsis mediterranei S699 ΔrifT was constructed by deleting the rifT gene. From this strain, eleven 8-deoxy-rifamycin derivatives (1-11) and seven known analogues (12-18) were isolated. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Compound 1 is a novel amide N-glycoside of seco-rifamycin. Compounds 2 and 3 feature conserved 11,12-seco-rifamycin W skeleton. The diverse post-modifications in the polyketide chain led to the production of 4-11. Compounds 2, 3, 5, 6, 13 and 15 exhibited antibacterial activity against Staphylococcus aureus (MIC (minimal inhibitory concentration) values of 10, 20, 20, 20, 40 and 20 µg/mL, respectively). Compounds 14, 15, 16, 17 and 18 showed potent antiproliferative activity against KG1 cells with IC50 (half maximal inhibitory concentration) values of 14.91, 44.78, 2.16, 18.67 and 8.07 µM, respectively.