Novel Skeletal Rearrangements of the Tigliane Diterpenoid Core.

To investigate the role of the secondary 5-hydroxy group in the activity of the anticancer drug tigilanol tiglate (2b) (Stelfonta), oxidation of this epoxytigliane diterpenoid from the Australian rainforest plant Fontainea picrosperma was attempted. Eventually, 5-dehydrotigilanol tiglate (3a) proved too unstable to be characterized in terms of biological activity and, therefore, was not a suitable tool compound for bioactivity studies. On the other hand, a series of remarkable skeletal rearrangements associated with the presence of a 5-keto group were discovered during its synthesis, including a dismutative ring expansion of ring A and a mechanistically unprecedented dyotropic substituent swap around the C-4/C-10 bond. Taken together, these observations highlight the propensity of the α-hydroxy-ß-diketone system to trigger complex skeletal rearrangements and pave the way to new areas of the natural products chemical space.

Identification of a Novel Curcumin Derivative Influencing Notch Pathway and DNA Damage as a Potential Therapeutic Agent in T-ALL.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy considered curable by modern clinical management. Nevertheless, the prognosis for T-ALL high-risk cases or patients with relapsed and refractory disease is still dismal. Therefore, there is a keen interest in developing more efficient and less toxic therapeutic approaches. T-ALL pathogenesis is associated with Notch signaling alterations, making this pathway a highly promising target in the fight against T-ALL. Here, by exploring the anti-leukemic capacity of the natural polyphenol curcumin and its derivatives, we found that curcumin exposure impacts T-ALL cell line viability and decreases Notch signaling in a dose- and time-dependent fashion. However, our findings indicated that curcumin-mediated cell outcomes did not depend exclusively on Notch signaling inhibition, but might be mainly related to compound-induced DNA-damage-associated cell death. Furthermore, we identified a novel curcumin-based compound named CD2066, endowed with potentiated anti-proliferative activity in T-ALL compared to the parent molecule curcumin. At nanomolar concentrations, CD2066 antagonized Notch signaling, favored DNA damage, and acted synergistically with the CDK1 inhibitor Ro3306 in T-ALL cells, thus representing a promising novel candidate for developing therapeutic agents against Notch-dependent T-ALL.

Curcumin-based-fluorescent probes targeting ALDH1A3 as a promising tool for glioblastoma precision surgery and early diagnosis.

Glioblastoma (GBM) is the most aggressive primary brain tumour for which both effective treatments and efficient tools for an early-stage diagnosis are lacking. Herein, we present curcumin-based fluorescent probes that are able to bind to aldehyde dehydrogenase 1A3 (ALDH1A3), an enzyme overexpressed in glioma stem cells (GSCs) and associated with stemness and invasiveness of GBM. Two compounds are selective versus ALDH1A3, without showing any appreciable interaction with other ALDH1A isoenzymes. Indeed, their fluorescent signal is detectable only in our positive controls in vitro and absent in cells that lack ALDH1A3. Remarkably, in vivo, our Probe selectively accumulate in glioblastoma cells, allowing the identification of the growing tumour mass. The significant specificity of our compounds is the necessary premise for their further development into glioblastoma cells detecting probes to be possibly used during neurosurgical operations.

Drug affinity-responsive target stability unveils filamins as biological targets for artemetin, an anti-cancer flavonoid.

Artemetin is a valuable 5-hydroxy-3,6,7,3',4'-pentamethoxyflavone present in many different medicinal plants with very good oral bioavailability and drug-likeness values, owing to numerous bioactivities, such as anti-inflammatory and anti-cancer ones. Here, a multi-disciplinary plan has been settled and applied for identifying the artemetin target(s) to inspect its mechanism of action, based on drug affinity-responsive target stability and targeted limited proteolysis. Both approaches point to the disclosure of filamins A and B as direct artemetin targets in HeLa cell lysates, also giving detailed insights into the ligand/protein-binding sites. Interestingly, also 8-prenyl-artemetin, which is an artemetin more permeable semisynthetic analog, directly interacts with filamins A and B. Both compounds alter filamin conformation in living HeLa cells with an effect on cytoskeleton disassembly and on the disorganization of the F-actin filaments. Both the natural compound and its derivative are able to block cell migration, expectantly acting on tumor metastasis occurrence and development.

Rotational constriction of curcuminoids impacts 5-lipoxygenase and mPGES-1 inhibition and evokes a lipid mediator class switch in macrophages.

Polypharmacological targeting of lipid mediator networks offers potential for efficient and safe anti-inflammatory therapy. Because of the diversity of its biological targets, curcumin (1a) has been viewed as a privileged structure for bioactivity or, alternatively, as a pan-assay interference (PAIN) compound. Curcumin has actually few high-affinity targets, the most remarkable ones being 5-lipoxygenase (5-LOX) and microsomal prostaglandin E2 synthase (mPGES)-1. These enzymes are critical for the production of pro-inflammatory leukotrienes and prostaglandin (PG)E2, and previous structure-activity-relationship studies in this area have focused on the enolized 1,3-diketone motif, the alkyl-linker and the aryl-moieties, neglecting the rotational state of curcumin, which can adopt twisted conformations in solution and at target sites. To explore how the conformation of curcuminoids impacts 5-LOX and mPGES-1 inhibition, we have synthesized rotationally constrained analogues of the natural product and its pyrazole analogue by alkylation of the linker and/or of the ortho aromatic position(s). These modifications strongly impacted 5-LOX and mPGES-1 inhibition and their systematic analysis led to the identification of potent and selective 5-LOX (3b, IC50 = 0.038 µM, 44.7-fold selectivity over mPGES-1) and mPGES-1 inhibitors (2f, IC50 = 0.11 µM, 4.6-fold selectivity over 5-LOX). Molecular docking experiments suggest that the C2-methylated pyrazolocurcuminoid 3b targets an allosteric binding site at the interface between catalytic and regulatory 5-LOX domain, while the o, o'-dimethylated desmethoxycurcumin 2f likely binds between two monomers of the trimeric mPGES-1 structure. Both compounds trigger a lipid mediator class switch from pro-inflammatory leukotrienes to PG and specialized pro-resolving lipid mediators in activated human macrophages.

The synthetic triterpenoids CDDO-TFEA and CDDO-Me, but not CDDO, promote nuclear exclusion of BACH1 impairing its activity.

The transcription factor BACH1 is a potential therapeutic target for a variety of chronic conditions linked to oxidative stress and inflammation, as well as cancer metastasis. However, only a few BACH1 degraders/inhibitors have been described. BACH1 is a transcriptional repressor of heme oxygenase 1 (HMOX1), which is positively regulated by transcription factor NRF2 and is highly inducible by derivatives of the synthetic oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO). Most of the therapeutic activities of these compounds are due to their anti-inflammatory and antioxidant properties, which are widely attributed to their ability to activate NRF2. However, with such a broad range of action, these compounds have other molecular targets that have not been fully identified and could also be of importance for their therapeutic profile. Herein we identified BACH1 as a target of two CDDO-derivatives (CDDO-Me and CDDO-TFEA), but not of CDDO. While both CDDO and CDDO-derivatives activate NRF2 similarly, only CDDO-Me and CDDO-TFEA inhibit BACH1, which explains the much higher potency of these CDDO-derivatives as HMOX1 inducers compared with unmodified CDDO. Notably, we demonstrate that CDDO-Me and CDDO-TFEA inhibit BACH1 via a novel mechanism that reduces BACH1 nuclear levels while accumulating its cytoplasmic form. In an in vitro model, both CDDO-derivatives impaired lung cancer cell invasion in a BACH1-dependent and NRF2-independent manner, while CDDO was inactive. Altogether, our study identifies CDDO-Me and CDDO-TFEA as dual KEAP1/BACH1 inhibitors, providing a rationale for further therapeutic uses of these drugs.

The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate.

SNAP-tag ® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a chemo-enzymatic approach, by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP-tag ® and its relative thermostable version (SsOGT-H5 ) proved to be very active on this substrate. The stability of these tags upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling protein-tag.

Identification of a Strigoterpenoid with Dual Nrf2 and Nf-κB Modulatory Activity.

The sesquiterpene-coumarin ether samarcandone provided a suitable framework to replace the apocarotenoid A-C ring system of strigol (1), replicating, after linking to a butenolide moiety, the activity of the natural phytohormone on Nrf2 and also showing potent NF-kB inhibitory activity, overall modulating two critical pathways of inflammation and cancer.

Chemoproteomic fishing identifies arzanol as a positive modulator of brain glycogen phosphorylase.

The interactome of arzanol was investigated by MS-based chemical proteomics, a pioneering technology for small molecule target discovery. Brain glycogen phosphorylase (bGP), a key regulator of glucose metabolism so far refractory to small molecule modulation, was identified as the main high-affinity target of arzanol. Competitive affinity-based proteomics, DARTS, molecular docking, surface plasmon resonance and in vitro biological assays provided molecular mechanistic insights into the arzanol-enzyme interaction, qualifying this positive modulator of bGP for further studies in the realm of neurodegeneration and cancer.

Cannabis Phenolics and their Bioactivities.

BACKGROUND: Although Cannabis sativa L. is one of the most versatile plant species with multipurpose use both as medical, alimentary source and as psychoactive abuse, its biomedical relevance focused the attention on major cannabinoids. Phytochemical characterization of cannabis highlights the presence of various non-cannabinoids constituents including flavonoids, spiroindans, dihyrostilbenes, dihydrophenanthrenes, lignanamides, steroids and alkaloids. This review aims to identify polyphenols present in this plant, their biosynthesis, their bioactivities and their synthesis, when this occurred. METHODS: We undertook a systematic research focused on bibliographic databases including all noncannabinoids phenolics in various C. sativa strains from their isolation, structural elucidation, their biological activity to their synthesis. RESULT: Nevertheless, attention has so far been focused only on cannabinoids (more than one hundred isolated), cannabis is a complex plant able to produce more than 480 chemical entities that represent almost all of the different biogenetic classes. Regarding phenolic compounds, the plant biosynthesises a plethora of unique non-cannabinoids second metabolites, such as prenylated flavonoids, stilbenoids derivatives and lignanammides. CONCLUSION: Cannabis is a plant with high pharmacological and nutrition values, its potentialities and applications are not only circumscribed to cannabinoids biological activities, but also defined by noncannabinoid compounds. The combination of other cannabinoids together with noncannabinoid components could enhance the beneficial effects of THC and could reduce undesirable side effects.

The reaction of cinnamaldehyde and cinnam(o)yl derivatives with thiols.

Spurred by the alleged relevance of the thia-Michael reaction in the bioactivity of various classes of cinnam(o)yl natural products and by the development of a quick NMR assay to study this reaction, we have carried out a systematic study of the "native" reactivity of these compounds with dodecanethiol and cysteamine as models, respectively, of simple thiols and reactive protein thiols that can benefit from iminium ion catalysis in Michael reactions. Cinnamoyl esters and amides, as well as cinnamyl ketones and oximes, did not show any reactivity with the two probe thiols, while cinnamaldehyde (1a) reacted with cysteamine to afford a mixture of a thiazoline derivative and compounds of multiple addition, and with aliphatic thiols to give a single bis-dithioacetal (6). Chalchones and their vinylogous C5-curcuminoid derivatives were the only cinnamoyl derivatives that gave a thia-Michael reaction. From a mechanistic standpoint, loss of conjugation in the adduct might underlie the lack of a native Michael reactivity. This property is restored by the presence of another conjugating group on the carbonyl, as in chalcones and C5-curcuminoids. A critical mechanistic revision of the chemical and biomedical literature on cinnamaldehyde and related compounds seems therefore required.

Celecoxib inhibits proliferation and survival of chronic myelogeous leukemia (CML) cells via AMPK-dependent regulation of ß-catenin and mTORC1/2.

CML is effectively treated with tyrosine kinase inhibitors (TKIs). However, the efficacy of these drugs is confined to the chronic phase of the disease and development of resistance to TKIs remains a pressing issue. The anti-inflammatory COX2 inhibitor celecoxib has been utilized as anti-tumour drug due to its anti-proliferative activity. However, its effects in hematological malignancies, in particular CML, have not been investigated yet. Thus, we tested biological effects and mechanisms of action of celecoxib in Philadelphia-positive (Ph+) CML and ALL cells.We show here that celecoxib suppresses the growth of Ph+ cell lines by increasing G1-phase and apoptotic cells and reducing S- and G2-phase cells. These effects were independent of COX2 inhibition but required the rapid activation of AMP-activated protein kinase (AMPK) and the consequent inhibition mTORC1 and 2. Treatment with celecoxib also restored GSK3ß function and led to down-regulation of ß-catenin activity through transcriptional and post-translational mechanisms, two effects likely to contribute to Ph+ cell growth suppression by celecoxib.Celecoxib inhibited colony formation of TKI-resistant Ph+ cell lines including those with the T315I BCR-ABL mutation and acted synergistically with imatinib in suppressing colony formation of TKI-sensitive Ph+ cell lines. Finally, it suppressed colony formation of CD34+ cells from CML patients, while sparing most CD34+ progenitors from healthy donors, and induced apoptosis of primary Ph+ ALL cells.Together, these findings indicate that celecoxib may serve as a COX2-independent lead compound to simultaneously target the mTOR and ß-catenin pathways, key players in the resistance of CML stem cells to TKIs.

Triazole-curcuminoids: A new class of derivatives for 'tuning' curcumin bioactivities?

Curcumin is a unique blend of pharmacophores responsible for the pleiotropy of this natural pigment. In the present study we have replaced the 1,3-dicarbonyl moiety with a 1,2,3-triazole ring to furnish a new class of triazole-curcuminoids as a possible strategy to generate new compounds with different potency and selectivity compared to curcumin. We obtained a proof-of-principle library of 28 compounds tested for their cytotoxicity (SY-SY5Y and HeLa cells) and for their ability to inhibit NF-κB. Furthermore, we also generated 1,3-dicarbonyl curcuminoids of selected click compounds. Triazole-curcuminoids lost their ability to be Michael's acceptors, yet maintained some of the features of the parent compounds and disclosed new ones. In particular, we found that some compounds were able to inhibit NF-κB without showing cytotoxicity, while others, unlike curcumin, activated NF-κB signalling. This validates the hypothesis that click libraries can be used to investigate the biological activities of curcumin as well as generate analogs with selected features.

SAR studies on curcumin's pro-inflammatory targets: discovery of prenylated pyrazolocurcuminoids as potent and selective novel inhibitors of 5-lipoxygenase.

The anticarcinogenic and anti-inflammatory properties of curcumin have been extensively investigated, identifying prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), key enzymes linking inflammation with cancer, as high affinity targets. A comparative structure-activity study revealed three modifications dissecting mPGES-1/5-LO inhibition, namely (i) truncation of the acidic, enolized dicarbonyl moiety and/or replacement by pyrazole, (ii) hydrogenation of the interaryl linker, and (iii) (dihydro)prenylation. The prenylated pyrazole analogue 11 selectively inhibited 5-LO, outperforming curcumin by a factor of up to 50, and impaired zymosan-induced mouse peritonitis along with reduced 5-LO product levels. Other pro-inflammatory targets of curcumin (i.e., mPGES-1, cyclooxygenases, 12/15-LOs, nuclear factor-κB, nuclear factor-erythroid 2-related factor-2, and signal transducer and activator of transcription 3) were hardly affected by 11. The strict structural requirements for mPGES-1 and 5-LO inhibition strongly suggest that specific interactions rather than redox or membrane effects underlie the inhibition of mPGES-1 and 5-LO by curcumin.

Functionalization of ß-caryophyllene generates novel polypharmacology in the endocannabinoid system.

The widespread dietary plant sesquiterpene hydrocarbon ß-caryophyllene (1) is a CB2 cannabinoid receptor-specific agonist showing anti-inflammatory and analgesic effects in vivo. Structural insights into the pharmacophore of this hydrocarbon, which lacks functional groups other than double bonds, are missing. A structure-activity study provided evidence for the existence of a well-defined sesquiterpene hydrocarbon binding site in CB2 receptors, highlighting its exquisite sensitivity to modifications of the strained endocyclic double bond of 1. While most changes on this element were detrimental for activity, ring-opening cross metathesis of 1 with ethyl acrylate followed by amide functionalization generated a series of new monocyclic amides (11a, 11b, 11c) that not only retained the CB2 receptor functional agonism of 1 but also reversibly inhibited fatty acid amide hydrolase (FAAH), the major endocannabinoid degrading enzyme, without affecting monoacylglycerol lipase (MAGL) and α,ß hydrolases 6 and 12. Intriguingly, further modification of this monocyclic scaffold generated the FAAH- and endocannabinoid substrate-specific cyclooxygenase-2 (COX-2) dual inhibitors 11e and 11f, which are probes with a novel pharmacological profile. Our study shows that by removing the conformational constraints induced by the medium-sized ring and by introducing functional groups in the sesquiterpene hydrocarbon 1, a new scaffold with pronounced polypharmacological features within the endocannabinoid system could be generated. The structural and functional repertoire of cannabimimetics and their yet poorly understood intrinsic promiscuity may be exploited to generate novel probes and ultimately more effective drugs.

Synthesis and tubulin-binding properties of non-symmetrical click C5-curcuminoids.

A click-type entry into shortened curcuminoids of the diarylpentanoid type has been developed. The reaction is ideally suited to generate non-symmetrical analogues of curcumin, a class of natural products difficult to access but of growing biomedical relevance and special mechanistic interest to investigate the unique binding mode of curcumin to tubulin. Investigation of a series of click diarylpentane curcuminoids and their pyrazole adducts in various cellular tubulin functional assays validated this class of compounds as a novel type of anti-mitotic agents, evidencing structure-activity relationships, and identifying the pyrazole adduct 4k as a promising lead.

Targeting oncogenic serine/threonine-protein kinase BRAF in cancer cells inhibits angiogenesis and abrogates hypoxia.

Carcinomas are comprised of transformed epithelial cells that are supported in their growth by a dedicated neovasculature. How the genetic milieu of the epithelial compartment influences tumor angiogenesis is largely unexplored. Drugs targeted to mutant cancer genes may act not only on tumor cells but also, directly or indirectly, on the surrounding stroma. We investigated the role of the BRAF(V600E) oncogene in tumor/vessel crosstalk and analyzed the effect of the BRAF inhibitor PLX4720 on tumor angiogenesis. Knock-in of the BRAF(V600E) allele into the genome of human epithelial cells triggered their angiogenic response. In cancer cells harboring oncogenic BRAF, the inhibitor PLX4720 switches off the ERK pathway and inhibits the expression of proangiogenic molecules. In tumor xenografts harboring the BRAF(V600E), PLX4720 extensively modifies the vascular network causing abrogation of hypoxia. Overall, our results provide a functional link between oncogenic BRAF and angiogenesis. Furthermore, they indicate how the tumor vasculature can be "indirectly" besieged through targeting of a genetic lesion to which the cancer cells are addicted.

Umbellulone modulates TRP channels.

Inhalation of umbellulone (UMB), the offensive principle of the so-called "headache tree" (California bay laurel, Umbellularia californica Nutt.), causes a painful cold sensation. We therefore studied the action of UMB and some derivatives devoid of thiol-trapping properties on the "cold" transient receptor potential cation channels TRPA1 and TRPM8. UMB activated TRPA1 in a dose-dependent manner that was attenuated by cysteine-to-serine isosteric mutation in TRPA1 (C622S), while channel block was observed at higher concentration. However, although activation by mustard oil was completely prevented in these mutants, UMB still retained activating properties, indicating that it acts on TRPA1 only as a partial electrophilic agonist. UMB also activated TRPM8, but to a lower extent than TRPA1. Removing Michael acceptor properties of UMB (reduction or nucleophilic trapping) was detrimental for the activation of TRPA1, but increased the blocking potency. This was, however, attenuated by acetylation of the hydroxylated analogs. All UMB derivatives, except the acetylated derivatives, were also TRPM8 activators. They acted, however, in a bimodal manner, inhibiting the channel more potently than UMB, and with tetrahydro-UMB being the most potent TRPM8 activator. In conclusion, UMB is a bimodal activator of TRPA1 and a weak activator of TRPM8. Non-electrophilic derivatives of UMB are better TRPM8 activators than the natural product and also potent blockers of this channel as well as of TRPA1. The lack of effects of the acetylated UMB derivatives suggests that steric hindrance may prevent access to the recognition site for the bicyclic monoterpene pharmacophore on TRPA1 and TRPM8.

Anti-inflammatory and vascularprotective properties of 8-prenylapigenin.

Flavonoids display several biological activities, but exhibit poor oral absorption and rapid metabolism. To improve their pharmacological profile four C8-prenyl flavonoids, structurally related to the anti-inflammatory lead apigenin, were synthesized, and the two least cytotoxic (IC(50)>30 microM) compounds [8-prenylnaringenin (8-PN) and 8-prenylapigenin (8-PA)] in RAW 264.7 murine macrophages were assayed against a panel of biological targets. The anti-inflammatory properties of these compounds were evaluated in an in vitro model of inflammation [cells exposed to 0.1 microg/ml lipopolysaccharide (LPS) for 24h]. Both 8-PN and 8-PA were equally effective and potent in inhibiting the LPS-induced gene expression [tumor necrosis factor (TNF)-alpha, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2] (RT-PCR) and release (ELISA) of pro-inflammatory mediators [TNF-alpha, NO, prostaglandin (PG)E(2)], through mechanisms involving the inhibition of nuclear factor-kappaB (NF-kappaB) activation (EMSA) and reactive oxygen species accumulation [2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) determination]. One-digit nM concentrations of 8-PN or 8-PA induced a significant increase in the basal production of the atheroprotective prostacyclin (PGI(2)) by human umbilical vein endothelial cells (HUVEC), with maximal effects at 10 nM. Both NS-398, a specific COX-2 inhibitor, and ICI 182 780, a non-selective estrogen receptor antagonist, abolished the activity of these compounds, suggesting a COX- and estrogen receptor-dependent mechanism of activity. 8-PA, a weaker estrogenic compound than 8-PN, resulted only 2-fold less potent than 8-PN in potentiating PGI(2) production by HUVEC, qualifying this C8-prenyl flavonoid as a lead for the rational design of new anti-inflammatory and vascularprotective compounds.