Lambertellin from Pycnoporus sanguineus MUCL 51321 and its anti-inflammatory effect via modulation of MAPK and NF-κB signaling pathways

Lambertellin (1) and ergosta-5,7,22-trien-3-ol (2) were isolated from the solid rice fermentation of the plant pathogenic fungus Pycnoporus sanguineus MUCL 51321. Their structures were elucidated using comprehensive spectroscopic methods. The isolated compounds were tested on lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. Lambertellin (1) exhibited promising inhibitory activity against nitric oxide (NO) production with IC₅₀ value of 3.19 µM, and it significantly inhibited the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2). Lambertellin (1) also decreased the expression of pro-inflammatory cytokines IL-6 and IL-1β. The study of the mechanistic pathways revealed that lambertellin (1) exerts its anti-inflammatory effect in LPS-stimulated RAW 264.7 macrophage cells by modulating the activation of the mitogen activated protein kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways. Therefore, lambertellin (1) could be a promising lead compound for the development of new anti-inflammatory drugs.     

Discovery of aromatic amides with triazole-core as potent reversal agents against P-glycoprotein-mediated multidrug resistance

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a major impediment for clinical cancer therapy. 19 novel aromatic amides with triazole-core as MDR reversal agents were designed and synthesized via click chemistry to reverse MDR. Among them, compound 42 was identified as the most promising candidate with high potency (EC₅₀ = 78.1 ± 5.4 nM), low cytotoxity (SI > 1282) and persistent duration in reversing doxorubicin (DOX) resistance in K562/A02 cells. 42 also enhanced the potency of other P-gp associated cytotoxic agents with different structures. In further study, remarkably increased intracellular accumulation of Rh123 and DOX in K562/A02 cells was achieved by compound 42, while CYP3A4 activity had no change by compound 42. These results indicate that compound 42 as a relatively safe modulator of P-gp-mediated MDR has good potential for further development.     

Exploring the antimalarial potential of the methoxy-thiazinoquinone scaffold: Identification of a new lead candidate

A small library of antiplasmodial methoxy-thiazinoquinones, rationally designed on the model of the previously identified hit 1, has been prepared by a simple and inexpensive procedure. The synthetic derivatives have been subjected to in vitro pharmacological screening, including antiplasmodial and toxicity assays. These studies afforded a new lead candidate, compound 9, endowed with higher antiplasmodial potency compared to 1, a good selectivity index when tested against a panel of mammalian cells, no toxicity against RBCs, a synergistic antiplasmodial action in combination with dihydroartemisinin, and a promising inhibitory activity on stage V gametocyte growth. Computational studies provided useful insights into the structural requirements needed for the antiplasmodial activity of thiazinoquinone compounds and on their putative mechanism of action.     

Discovery of amide-bridged pyrrolo[2,3-d]pyrimidines as tumor targeted classical antifolates with selective uptake by folate receptor α and inhibition of de novo purine nucleotide biosynthesis

We previously showed that classical 6-substituted pyrrolo[2,3-d]pyrimidine antifolates bind to folate receptor (FR) α and the target purine biosynthetic enzyme glycinamide ribonucleotide formyltransferase (GARFTase) with different cis and trans conformations. In this study, we designed novel analogs of this series with an amide moiety in the bridge region that can adopt both the cis and trans lowest energy conformations. This provides entropic benefit, by restricting the number of side-chain conformations of the unbound ligand to those most likely to promote binding to FRα and the target enzyme required for antitumor activity. NMR of the most active compound 7 showed both cis and trans amide bridge conformations in ~1:1 ratio. The bridge amide group in the best docked poses of 7 in the crystal structures of FRα and GARFTase adopted both cis and trans conformations, with the lowest energy conformations predicted by Maestro and evidenced by NMR within 1 kcal/mol. Compound 7 showed ~3-fold increased inhibition of FRα-expressing cells over its non-restricted parent analog 1 and was selectively internalized by FRα over the reduced folate carrier (RFC), resulting in significant in vitro antitumor activity toward FRα-expressing KB human tumor cells. Antitumor activity of 7 was abolished by treating cells with adenosine but was incompletely protected by 5-aminoimidazole-4-carboxamide (AICA) at higher drug concentrations, suggesting GARFTase and AICA ribonucleotide formyltransferase (AICARFTase) in de novo purine biosynthesis as the likely intracellular targets. GARFTase inhibition by compound 7 was confirmed by an in situ cell-based activity assay. Our results identify a “first-in-class” classical antifolate with a novel amide linkage between the scaffold and the side chain aryl L-glutamate that affords exclusive selectivity for transport via FRα over RFC and antitumor activity resulting from inhibition of GARFTase and likely AICARFTase. Compound 7 offers significant advantages over clinically used inhibitors of this class that are transported by the ubiquitous RFC, resulting in dose-limiting toxicities.     

Steroids from Ganoderma sinense as new natural inhibitors of cancer-associated mutant IDH1

Isocitrate dehydrogenase (IDH) is one of the key enzymes in the tricarboxylic acid cycle, and IDH mutations have been associated with many cancers, including glioblastoma, sarcoma, acute myeloid leukemia, etc. Three natural steroids 1–3 from Ganoderma sinense, a unique and rare edible-medicinal fungi in China, were found as potential IDH1 inhibitors by virtual ligand screening method. Among the three compounds, 3 showed the highest binding affinity to IDH1 with significant calculated binding free energy. Enzymatic kinetics demonstrated that 3 inhibited mutant enzyme in a noncompetitive manner. The half effective concentration of 3 for reducing the concentration of D-2HG in HT1080 cells was 35.97 μM. The levels of histone H3K9me3 methylation in HT1080 cells were reduced by treating with 3. Furthermore, knockdown of mutant IDH1 in HT1080 cells decreased the anti-proliferative sensitivity to 3. In short, our findings highlight that compound 3 may have clinical potential in tumor therapies as an effective inhibitor of mutant IDH1.     

Novel inhibitors of leukocyte transendothelial migration

Leukocyte transendothelial migration is one of the most important step in launching an inflammatory immune response and chronic inflammation can lead to devastating diseases. Leukocyte migration inhibitors are considered as promising and potentially effective therapeutic agents to treat inflammatory and auto-immune disorders. In this study, based on previous trioxotetrahydropyrimidin based integrin inhibitors that suboptimally blocked leukocyte adhesion, twelve molecules with a modified scaffold were designed, synthesized, and tested in vitro for their capacity to block the transendothelial migration of immune cells. One of the molecules, namely, methyl 4-((2-(tert-butyl)-6-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene) methyl) phenoxy) methyl) benzoate, (compound 12), completely blocked leukocyte transendothelial migration, without any toxic effects on immune or endothelial cells (IC₅₀ = 2.4 µM). In vivo, compound 12 exhibited significant therapeutic effects in inflammatory bowel disease (IBD)/Crohn’s disease, multiple sclerosis, fatty liver disease, and rheumatoid arthritis models. A detailed acute and chronic toxicity profile of the lead compound in vivo did not reveal any toxic effects. Such a type of molecule might therefore provide a unique starting point for designing a novel class of leukocyte transmigration blocking agents with broad therapeutic applications in inflammatory and auto-immune pathologies.     

Novel steroidal 1,3,4-thiadiazines: Synthesis and biological evaluation in androgen receptor-positive prostate cancer 22Rv1 cells

A flexible approach to previously unknown spirofused and linked 1,3,4-thiadiazine derivatives of steroids with selective control of heterocyclization patterns is disclosed. (N-Arylcarbamoyl)spiroandrostene-17,6′ [1,3,4]thiadiazines and (N-arylcarbamoyl)17-[1′,3′,4′]thiadiazine-substituted androstenes, novel types of heterosteroids, were prepared from 16β,17β-epoxypregnenolone and 21-bromopregna-5,16-dien-20-one in good to high yields by the treatment with oxamic acid thiohydrazides. The synthesized compounds were screened for antiproliferative activity against the human androgen receptor-positive prostate cancer cell line 22Rv1. Most of (N-arylcarbamoyl)17-[1′,3′,4′]thiadiazine-substituted androstenes exhibit better antiproliferative potency (IC₅₀ = 2.1–6.6 µM) than the antiandrogen bicalutamide. Compounds 7d with IC₅₀ = 3.0 μM and 7j with IC₅₀ = 2.1 μM proved to be the most active in the series under study. Lead synthesized compound 7j downregulates AR expression and activity in 22Rv1 cells. NF-κB activity is also blocked in 7j-treated 22Rv1 cells. Apoptosis is considered as a possible mechanism of 7j-induced cell death.     

Exploiting the 7-methylimidazo[1,5-a]pyrazin-8(7H)-one scaffold for the development of novel chemical inhibitors for Bromodomain and Extraterminal Domain (BET) family

The bromodomain and extraterminal (BET) family of proteins play a crucial role in promoting gene expression of critical oncogenes. Novel BET bromodomain inhibitors with excellent potency, drug metabolism and pharmacokinetics (DMPK) properties were in strong need for development. We reported a series of potential BET inhibitors through incorporation of imidazole into pyridine scaffold. Among them, a novel BET inhibitor with 7-methylimidazo[1,5-a]pyrazin-8(7H)-one core, compound 28, was considered to be the most promising for in-depth study. Compound 28 exhibited excellent BRD4-inhibitory activity with IC₅₀ value of 33 nM and anti-proliferation potency with IC₅₀ value of 110 nM in HL-60 (human promyelocytic leukemia) cancer cell lines. Western Blot indicated that compound 28 can effectively trigger apoptosis in BxPc3 cells by modulating the intrinsic apoptotic pathway. In conclusion, these results suggested that compound 28 has merely potential for leukemia treatment.     

Discovery of potent PTP1B inhibitors via structure-based drug design,synthesis and in vitro bioassay of Norathyriol derivatives

Protein tyrosine phosphatase 1B (PTP1B) has recently been identified as a potential target of Norathyriol. Unfortunately, Norathyriol is not a potent PTP1B inhibitor, which somewhat hinders its further application. Based on the fact that no study on the relationship of chemical structure and PTP1B inhibitory activity of Norathyriol has been reported so far, we attempted to perform structural optimization so as to improve the potency for PTP1B. Via structure-based drug design (SBDD), a rational strategy based on the binding mode of Norathyriol to PTP1B, we designed 26 derivatives with substitutions at the four phenolic hydroxyl groups of Norathyriol. By chemical synthesis and in vitro bioassay, we identified seven PTP1B inhibitors that were more potent than Norathyriol, of which XWJ24 showed the highest potency (IC₅₀: 0.6 μM). We also found out that XWJ24 was a competitive inhibitor and showed the 4.5-fold selectivity over its close homolog, TC-PTP. Through molecular docking of XWJ24 against PTP1B, we highlighted the essential role of its hydrogen bond with Asp181 for PTP1B inhibition and identified a potential halogen bond with Asp48 that was not observed for Norathyriol. The current data indicate that our SBDD strategy is effective to discover potent PTP1B-targeted Norathyriol derivatives, and XWJ24 is a promising lead compound for further development.     

Development of pyrazole and spiropyrazoline analogs as multifunctional agents for treatment of Alzheimer’s disease

Cholinergic hypothesis of Alzheimer’s disease has been advocated as an essential tool in the last couple of decades for the drug development. Here in, we report de novo fragment growing strategy for the design of novel 3,5-diarylpyrazoles and hit optimization of spiropyrazoline derivatives as acetyl cholinesterase inhibitors. Both type of scaffolds numbering forty compounds were synthesized and evaluated for their potencies against AChE, BuChE and PAMPA. Introduction of lipophilic cyclohexane ring in 3,5-diarylpyrazole analogs led to spiropyrazoline derivatives, which facilitated and improved the potencies. Compound 44 (AChE = 1.937 ± 0.066 µM; BuChE = 1.166 ± 0.088 µM; hAChE = 1.758 ± 0.095 µM; P_e = 9.491 ± 0.34 × 10⁻⁶ cm s¹) showed positive results, which on further optimization led to the development of compound 67 (AChE = 0.464 ± 0.166 µM; BuChE = 0.754 ± 0.121 µM; hAChE = 0.472 ± 0.042 µM; P_e = 13.92 ± 0.022 × 10⁻⁶ cm s¹). Compounds 44 and 67 produced significant displacement of propidium iodide from the peripheral anionic site (PAS) of AChE. They were found to be safer to MC65 cells and decreased metal induced Aβ_1-42 aggregation. Further, in-vivo behavioral studies, on scopolamine induced amnesia model, the compounds resulted in better percentage spontaneous alternation scores and were safe, had no influence on locomotion in tested animal groups at dose of 3 mg/kg. Early pharmacokinetic assessment of optimized hit molecules was supportive for further drug development.     

Identification of 10-dehydrooxyglycyuralin E as a selective human estrogen receptor alpha partial agonist

Selective estrogen receptor modulators (SERMs) act as either agonist or antagonist of estrogen receptor (ER) in a tissue selective manner and have been used in several diseases such as breast cancer, postmenopausal syndrome, osteoporosis, and cardiovascular diseases. However, current SERMs may also increase the risk of serious side effects and trigger drug resistance. Herein, a screening program, that was designed to search for novel SERMs, resulted in the identification of a series of 2-arylbenzofuran-containing compounds that are ligands for ERα, when applying the Gaussia-luciferase reporter assay. One of these compounds, 10-dehydrooxyglycyuralin E (T9) was chemically synthesized. T9 showed anti-estrogenic/proliferative activity in ERα-positive breast cancer cells. Pretreatment of T9 prevented the mRNA expression of GREB1, which is an estrogen response gene. Furthermore, by an in silico docking simulation study we demonstrated that T9 showed interactions directly to ERα. Taken together, these results demonstrated that T9 is a candidate of SERMs and a useful seed compound for the foundation of the selective activity of SERMs.     

Oxadiazolone derivatives,new promising multi-target inhibitors against M. tuberculosis

A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc²6230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC₅₀, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.     

Pentraxin 3 is an anti-inflammatory protein associated with lipid-induced interleukin 10 in vitro

Pentraxin 3 (PTX3) is an acute phase protein expressed in response to pro-inflammatory stimuli during atherosclerosis. However, recent findings suggest that PTX3 is a counter-regulatory protein which enhances the anti-inflammatory response.ObjectiveTherefore, the capacity of PTX3 to alter the inflammatory milieu following in vitro stimulation of PBMCs with the pro-inflammatory lipid, palmitate, was examined.MethodsPBMCs from 17 healthy male donors were isolated and cultured under four separate conditions; 200 μmol/L palmitate, a physiologically relevant concentration of PTX3, in combination (pal + PTX3), and an unstimulated time-course control.ResultsPalmitate-induced production of the counter-regulatory protein PTX3 was positively associated with the production of the anti-inflammatory cytokine interleukin 10 (IL-10) following in vitro stimulation of human PBMCs. Furthermore, stimulation of PBMCs in vitro with 500 pg/mL PTX3 elicited a significantly greater increase in IL-10 production compared to the palmitate stimulated conditions. However, PTX3 stimulation did not result in the production of the pro-inflammatory cytokines IL-1β, IL-6, and tumor necrosis factor alpha, and when combined with palmitate, did not alter the pro-inflammatory milieu from PBMCs in this study.ConclusionThese findings provide evidence supporting the role of PTX3 as a mediator of the anti-inflammatory response in physiologically relevant conditions, and suggests that PTX3 counter regulates the development of atherosclerosis by enhancing the production of IL-10.     

Unveiling novel diphenyl-1H-pyrazole based acrylates tethered to 1,2,3-triazole as promising apoptosis inducing cytotoxic and anti-inflammatory agents

Meagre and suboptimal therapeutic response along with the side effect profile associated with the existing anticancer therapy have necessitated the development of new therapeutic modalities to curb this disease. Bearing in mind the current scenario, a series of 1,2,3-triazole linked 3-(1,3-diphenyl-1H-pyrazol-4-yl)acrylates was synthesized following a multi-step reaction scheme. Initial screening for anticancer potential was done by in vitro sulforhodamine B assay against four human cancer cell lines- MCF-7 (breast), A549 (Lung) and HCT-116 and HT-29 (Colon). On evaluation, several compounds showed promising growth inhibition against all the cell lines, particularly compounds 6e, 6f and 6n. Among them, compound 6f displayed IC₅₀ values of 1.962, 3.597, 1.764 and 4.496 µM against A549, HCT-116, MCF-7 and HT-29 cell lines respectively. Furthermore, the apoptosis inducing potential of the compounds was determined by Hoechst staining and DNA fragmentation assay. Colony formation inhibition assay was also carried out to determine the long term cytotoxic potential of the molecules. Moreover, compounds 6e, 6f and 6n were also evaluated for anti-inflammatory activity by protein albumin denaturation assay and red blood cell membrane stabilizing assay.     

Novel quinazolin-4-one derivatives as potentiating agents of doxorubicin cytotoxicity

We report the design, synthesis and biological evaluation of 17 novel 8-aryl-2-morpholino-3,4-dihydroquinazoline derivatives based on the standard model of DNA-PK and PI3K inhibitors. Novel compounds are sub-divided into two series where the second series of five derivatives was designed to have a better solubility profile over the first one. A combination of in vitro and in silico techniques suggested a plausible synergistic effect with doxorubicin of the most potent compound 14d on cell proliferation via DNA-PK and poly(ADP-ribose) polymerase-1 (PARP-1) inhibition, while alone having a negligible effect on cell proliferation.     

The development of 2-acetylphenol-donepezil hybrids as multifunctional agents for the treatment of Alzheimer’s disease

A series of 2-acetylphenol-donepezil hybrids was designed and synthesized based on multi-target-directed ligands strategy. The biological activities were evaluated by AChE/BChE inhibition and MAO-A/MAO-B inhibition. The results revealed that the tertiary amines and methylene chain length significantly affected the eeAChE inhibitory potency, in particular, compound TM-14 showed the best eeAChE inhibitory activity with IC₅₀ value of 2.9 μM, in addition, both kinetic analysis of AChE inhibition and docking study displayed that TM-14 could simultaneously bind to the catalytic active site and peripheral anionic site of AChE. Moreover, compound TM-14 was a selective metal chelator and could form 1:1 TM-14-Cu²⁺ complex. The structure-active-relationship also indicated that the O-alkylamine fragment remarkably decreased hMAO-B inhibitory activity, compound TM-2 exhibited potent hMAO-B inhibitory activity (IC₅₀ = 6.8 μM), which was supported by the molecular docking study. More interestingly, compounds TM-14 and TM-2 could cross the blood-brain barrier in vitro. Therefore, the structure-active-relationship of 2-acetylphenol-donepezil hybrids could encourage the development of multifunction agents with selective AChE inhibition or selective MAO-B inhibition for the treatment of Alzheimer’s disease.     

Cannabinoid receptor signaling in progenitor/stem cell proliferation and differentiation

Cannabinoids, the active components of cannabis (Cannabis sativa) extracts, have attracted the attention of human civilizations for centuries, much earlier than the discovery and characterization of their substrate of action, the endocannabinoid system (ECS). The latter is an ensemble of endogenous lipids, their receptors [in particular type-1 (CB₁) and type-2 (CB₂) cannabinoid receptors] and metabolic enzymes. Cannabinoid signaling regulates cell proliferation, differentiation and survival, with different outcomes depending on the molecular targets and cellular context involved. Cannabinoid receptors are expressed and functional from the very early developmental stages, when they regulate embryonic and trophoblast stem cell survival and differentiation, and thus may affect the formation of manifold adult specialized tissues derived from the three different germ layers (ectoderm, mesoderm and endoderm). In the ectoderm-derived nervous system, both CB₁ and CB₂ receptors are present in neural progenitor/stem cells and control their self-renewal, proliferation and differentiation. CB₁ and CB₂ show opposite patterns of expression, the former increasing and the latter decreasing along neuronal differentiation. Recently, endocannabinoid (eCB) signaling has also been shown to regulate proliferation and differentiation of mesoderm-derived hematopoietic and mesenchymal stem cells, with a key role in determining the formation of several cell types in peripheral tissues, including blood cells, adipocytes, osteoblasts/osteoclasts and epithelial cells. Here, we will review these new findings, which unveil the involvement of eCB signaling in the regulation of progenitor/stem cell fate in the nervous system and in the periphery. The developmental regulation of cannabinoid receptor expression and cellular/subcellular localization, together with their role in progenitor/stem cell biology, may have important implications in human health and disease.     

Negative regulation of the hepatic fibrogenic response by suppressor of cytokine signaling 1

Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFNγ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFNγ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFNγ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in HSC activation. Liver fibrosis was induced in Socs1^−/−Ifng^−/− mice with dimethylnitrosamine or carbon tetrachloride. Ifng^−/− and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1^−/−Ifng^−/− mice showed elevated serum ALT levels and increased liver fibrosis compared to Ifng^−/− mice. The latter group showed higher ALT levels and fibrosis than C57BL/6 controls. The livers of SOCS1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. SOCS1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from SOCS1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of SOCS1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFNγ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability.