Tigilanol tiglate is an oncolytic small molecule that induces immunogenic cell death and enhances the response of both target and non-injected tumors to immune checkpoint blockade.

BACKGROUND: Tigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that intratumoral (I.T.) injection of TT causes vascular disruption with concomitant tumor ablation in several preclinical models of cancer, in addition to various (sub)cutaneous tumors presenting in the veterinary clinic. TT has completed Phase I dose escalation trials, with some patients showing signs of abscopal effects. However, the exact molecular details underpinning its mechanism of action (MoA), together with its immunotherapeutic potential in oncology remain unclear. METHODS: A combination of microscopy, luciferase assays, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, phagocytosis assays and mixed lymphocyte reactions were used to probe the MoA of TT in vitro. In vivo studies with TT used MM649 xenograft, CT-26 and immune checkpoint inhibitor refractory B16-F10-OVA tumor bearing mice, the latter with or without anti-programmed cell death 1 (PD-1)/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) mAb treatment. The effect of TT at injected and non-injected tumors was also assessed. RESULTS: Here, we show that TT induces the death of endothelial and cancer cells at therapeutically relevant concentrations via a caspase/gasdermin E-dependent pyroptopic pathway. At therapeutic doses, our data demonstrate that TT acts as a lipotoxin, binding to and promoting mitochondrial/endoplasmic reticulum (ER) dysfunction (leading to unfolded protein responsemt/ER upregulation) with subsequent ATP depletion, organelle swelling, caspase activation, gasdermin E cleavage and induction of terminal necrosis. Consistent with binding to ER membranes, we found that TT treatment promoted activation of the integrated stress response together with the release/externalization of damage-associated molecular patterns (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through vaccination and rechallenge experiments using CT-26 colon carcinoma tumor bearing mice. Furthermore, TT also reduced tumor volume, induced immune cell infiltration, as well as improved survival in B16-F10-OVA tumor bearing mice when combined with immune checkpoint blockade. CONCLUSIONS: These data demonstrate that TT is an oncolytic small molecule with multiple targets and confirms that cell death induced by this compound has the potential to augment antitumor responses to immunotherapy.

Comparative Evaluation of Anticancer Activity of Natural Methoxylated Flavones Xanthomicrol and Eupatilin in A375 Skin Melanoma Cells.

Melanoma is a skin cancer caused by the malignant transformation of melanocytes and cutaneous melanoma represents the most aggressive and deadliest type of skin cancer with an increasing incidence worldwide. The main purpose of the present research was to evaluate the anticancer effects of the natural bioactive compounds xanthomicrol (XAN) and eupatilin (EUP) in human A375 malignant skin melanoma cells, a cell line widely used as an in vitro model of cutaneous melanoma. XAN and EUP are lipophilic methoxylated flavones with antioxidant, anti-inflammatory, and antitumor properties. The effects of XAN and EUP on cell viability, morphology, lipid profile, oxidative status, apoptosis, and mitochondrial membrane polarization were determined and compared in A375 cells. At 24 h-incubation (MTT assay), XAN significantly reduced viability at the dose range of 2.5-200 µM, while EUP showed a significant cytotoxicity from 25 µM. Moreover, both methoxylated flavones induced (at 10 and 25 µM, 24 h-incubation) marked cell morphological alterations (presence of rounded and multi-nucleated cells), signs of apoptosis (NucView 488 assay), and a noteworthy mitochondrial membrane depolarization (MitoView 633 assay), coupled to a marked lipid profile modulation, including variations in the ratio of phospholipid/cholesterol and a decrease in the oleic, palmitic, and palmitoleic acid amounts. Moreover, a remarkable time-dependent ROS generation (2',7'-dichlorodihydrofluorescein diacetate assay) was observed during 3 h-incubation of A375 cancer cells in the presence of XAN and EUP (10 and 25 µM). Our results confirm the potential antitumor effect of natural EUP and XAN in cutaneous melanoma by the activation of multiple anticancer mechanisms.

Arzanol, a natural phloroglucinol α-pyrone, protects HaCaT keratinocytes against H2O2-induced oxidative stress, counteracting cytotoxicity, reactive oxygen species generation, apoptosis, and mitochondrial depolarization.

Skin oxidative stress results in structural damage, leading to premature senescence, and pathological conditions such as inflammation and cancer. The plant-derived prenylated pyrone-phloroglucinol heterodimer arzanol, isolated from Helichrysum italicum ssp. microphyllum (Willd.) Nyman aerial parts, exhibits anti-inflammatory, anticancer, antimicrobial, and antioxidant activities. This study explored the arzanol protection against hydrogen peroxide (H2O2) induced oxidative damage in HaCaT human keratinocytes in terms of its ability to counteract cytotoxicity, reactive oxygen species (ROS) generation, apoptosis, and mitochondrial membrane depolarization. Arzanol safety on HaCaT cells was preliminarily examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and microscopic observation. The arzanol pre-incubation (5-100 µM, for 24 h) did not induce cytotoxicity and morphological alterations. The phloroglucinol, at 50 µM, significantly protected keratinocytes against cytotoxicity induced by 2 h-incubation with 2.5 and 5 mM H2O2, decreased cell ROS production induced by 1 h-exposure to all tested H2O2 concentrations (0.5-5 mM), as determined by the 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) assay, and lipid peroxidation (thiobarbituric acid reactive substances [TBARS] method). The 2-h incubation of keratinocytes with H2O2 determined a significant increase of apoptotic cells versus control cells, evaluated by NucView® 488 assay, from the dose of 2.5 mM. Moreover, an evident mitochondrial membrane potential depolarization, monitored by fluorescent mitochondrial dye MitoView™ 633, was assessed at 5 mM H2O2. Arzanol pre-treatment (50 µM) exerted a strong significant protective effect against apoptosis, preserving the mitochondrial membrane potential of HaCaT cells at the highest H2O2 concentrations. Our results validate arzanol as an antioxidant agent for the prevention/treatment of skin oxidative-related disorders, qualifying its potential use for cosmeceutical and pharmaceutical applications.

PPARα/γ-Targeting Amorfrutin Phytocannabinoids from Aerial Parts of Glycyrrhiza foetida.

An LC-MS/MS-guided analysis of the aerial parts of Glycyrrhiza foetida afforded new phenethyl (amorfrutin)- and alkyl (cannabis)-type phytocannabinoids (six and four compounds, respectively). The structural diversity of the new amorfrutins was complemented by the isolation of six known members and the synthesis of analogues modified on the aralkyl moiety. All of the compounds so obtained were assayed for agonist activity on PPARα and PPARγ nuclear receptors. Amorfrutin A (1) showed the highest agonist activity on PPARγ, amorfrutin H (7) selectively targeted PPARα, and amorfrutin E (4) behaved as a dual agonist, with the pentyl analogue of amorfrutin A (11) being inactive. Decarboxyamorfrutin A (2) was cytotoxic, and modifying its phenethyl moiety to a styryl or a phenylethynyl group retained this trait, suggesting an alternative biological scenario for these compounds. The putative binding modes of amorfrutins toward PPARα and PPARγ were obtained by a combined approach of molecular docking and molecular dynamics simulations, which provided insights on the structure-activity relationships of this class of compounds.

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.

Cannabidiol acts as molecular switch in innate immune cells to promote the biosynthesis of inflammation-resolving lipid mediators.

Cannabinoids are phytochemicals from cannabis with anti-inflammatory actions in immune cells. Lipid mediators (LM), produced from polyunsaturated fatty acids (PUFA), are potent regulators of the immune response and impact all stages of inflammation. How cannabinoids influence LM biosynthetic networks is unknown. Here, we reveal cannabidiol (CBD) as a potent LM class-switching agent that stimulates the production of specialized pro-resolving mediators (SPMs) but suppresses pro-inflammatory eicosanoid biosynthesis. Detailed metabololipidomics analysis in human monocyte-derived macrophages showed that CBD (i) upregulates exotoxin-stimulated generation of SPMs, (ii) suppresses 5-lipoxygenase (LOX)-mediated leukotriene production, and (iii) strongly induces SPM and 12/15-LOX product formation in resting cells by stimulation of phospholipase A2-dependent PUFA release and through Ca2+-independent, allosteric 15-LOX-1 activation. Finally, in zymosan-induced murine peritonitis, CBD increased SPM and 12/15-LOX products and suppressed pro-inflammatory eicosanoid levels in vivo. Switching eicosanoid to SPM production is a plausible mode of action of CBD and a promising inflammation-resolving strategy.

Scalemic natural products.

Covering: up to the end of 2022The area of scalemic natural products is often enigmatic from a mechanistic standpoint, since low optical purity is observed in compounds having multiple contiguous stereogenic centers resulting from mechanistically distinct biogenetic steps. A scalemic state is rarely the result of a sloppy enzymatic activity, rather resulting from the expression of antipodal enzymes/directing proteins or from the erosion of optical purity by enzymatic or spontaneous reactions. Evidence for these processes is critically reviewed, identifying the mechanisms most often associated to the enzymatic generation of scalemic natural products and also discussing analytical exploitations of natural products' scalemicity.

Natural Cannabichromene (CBC) Shows Distinct Scalemicity Grades and Enantiomeric Dominance in Cannabis sativa Strains.

Cannabichromene (CBC, 1a) occurs in Cannabis (Cannabis sativa) as a scalemate having a composition that is strain-dependent in terms of both enantiomeric excess and enantiomeric dominance. In the present work, the chirality of CBC (1a), a noncrystalline compound, was shown not to be significantly affected by standard conditions of isolation and purification, and enantiomeric self-disproportionation effects were minimized by carrying out the chiral analysis on crude fractions rather than on purified products. A genetic basis for the different enantiomeric state of CBC in Cannabis therefore seems to exist, implying that the chirality status of natural CBC (1a) in the plant is associated with the differential expression of CBCA-synthase isoforms and/or of associated directing proteins with antipodal enantiospecificity. The biological profile of both enantiomers of CBC should therefore be investigated independently to assess the contribution of this compound to the activity of Cannabis preparations.

Structurally Diverse Diterpenoids from the Sanya Bay Nudibranch Hexabranchus sanguineus and Its Sponge-Prey Chelonaplysilla sp.

Investigation of the South China Sea nudibranch Hexabranchus sanguineus from Sanya Bay afforded, in addition to three known compounds, nine new diterpenoids of the 5,19-cycloclerodane- (sanyanolides A-D), clerodane- (sanyanolide E) and subersin- (sanyanolides F-I) type. Remarkably, six diterpenoids aforementioned from H. sanguineus were also isolated from the sponge Chelonaplysilla sp. from the same water region, suggesting a trophic relationship between H. sanguineus and Chelonaplysilla sp. The structure and absolute configuration of new compounds were established by a combination of spectroscopic data, X-ray diffraction analysis and/or time-dependent density functional theory/electronic circular dichroism calculations. A plausible biogenetic relationship between these diterpenoids, along with the chemo-ecological implications of their co-occurrence in the two organisms investigated, was proposed and discussed. In in vitro bioassays, echinoclerodane A exhibited a potent inhibitory effect (IC50 =2.81 µM) on LPS-induced inflammatory response in RAW 264.7 macrophage cells. In addition, echinoclerodane A and oculatolide showed considerable antibacterial activities with MIC values ranging from 1.0 to 8.0 µg/mL.

Non-Volatile Terpenoids and Lipophilic Flavonoids from Achillea erba-rotta Subsp. moschata (Wulfen) I. Richardson.

Musk yarrow (Achillea erba-rotta subsp. moschata (Wulfen) I. Richardson) is endemic to the Central Alps, and is used to flavour alcoholic beverages. Despite its popularity as aromatizing agent and its alleged beneficial effects on digestion, the phytochemical profile of the plant is still largely unknown and undiscovered. As a consequence, its authentication in aromatized products is impossible beyond sensory analysis allowing forgery. To address these issues, we phytochemically characterized a sample of musk yarrow from the Italian Eastern Alps, identifying, in addition to widespread phytochemicals (taraxasterol, apigenin), the guaianolides 3, 8, 9; the seco-caryophyllane 6; and the polymethoxylated lipophilic flavonoids 1, 4, and 5. The flavonoid xanthomicrol 1, a major constituent of the plant, was cytotoxic to HeLa cells, but only modestly affected primary 3T3 fibroblasts. On account of their stability, detectability by UV absorption, and concentration, the oxygenated flavonoids qualify as markers to validate the supply chain of the plant growers to consumers.

Minor Phytocannabinoids: A Misleading Name but a Promising Opportunity for Biomedical Research.

Despite the very large number of phytocannabinoids isolated from Cannabis (Cannabis sativa L.), bioactivity studies have long remained focused on the so called "Big Four" [Δ9-THC (1), CBD (2), CBG (3) and CBC (4)] because of their earlier characterization and relatively easy availability via isolation and/or synthesis. Bioactivity information on the chemical space associated with the remaining part of the cannabinome, a set of ca 150 compounds traditionally referred to as "minor phytocannabinoids", is scarce and patchy, yet promising in terms of pharmacological potential. According to their advancement stage, we sorted the bioactivity data available on these compounds, better referred to as the "dark cannabinome", into categories: discovery (in vitro phenotypical and biochemical assays), preclinical (animal models), and clinical. Strategies to overcome the availability issues associated with minor phytocannabinoids are discussed, as well as the still unmet challenges facing their development as mainstream drugs.

Cryptic Epoxytiglianes from the Kernels of the Blushwood Tree (Fontainea picrosperma).

The kernels of the Australian blushwood tree (Fontainea picrosperma) are the source of the veterinary anticancer drug tigilanol tiglate (2a, Stelfonta) and contain a concentration of phorboids significantly higher than croton oil, the only abundant source of these compounds previously known. The oily matrix of the blushwood kernels is composed of free fatty acids and not by glycerides as found in croton oil. By active partitioning, it was therefore possible to recover and characterize for the first time a cryptic tigliane fraction, that is, the diterpenoid fraction that, because of its lipophilicity, could not be obtained by solvent partition of crude extracts. The cryptic tigliane fraction accounted for ca. 30% of the tigliane kernel titer and was quantified by 1H NMR spectroscopy and profiled by HPLC-MS. Long-chain (linoleates and/or oleates) 20-acyl derivatives of the epoxytigliane diesters tigilanol tiglate (EBC-46, 2a), EBC-47 (4a), EBC-59 (5a), EBC-83 (6a), and EBC-177 (7a) were identified. By chemoselective acylation of EBC-46 (2a) and EBC-177 (7a) the natural triesters 2b and 7b and a selection of analogues were prepared to assist identification of the natural compounds. The presence of a free C-20 hydroxy group is a critical requirement for PKC activation by phorbol esters. The unexpected activity of 20-linoleoyl triester 2b in a cytotoxicity assay based on PKC activation was found to be related mainly to its hydrolysis to tigilanol tiglate (2a) under the prolonged conditions of the assay, while other esters were inactive. Significant differences between the esterification profile of the epoxytigliane di- and triesters exist in F. picrosperma, suggesting a precise, yet elusive, blueprint of acyl decoration for the tigliane polyol 5-hydroxyepoxyphorbol.

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.

Pyrazole-Curcumin Suppresses Cardiomyocyte Hypertrophy by Disrupting the CDK9/CyclinT1 Complex.

The intrinsic histone acetyltransferase (HAT), p300, has an important role in the development and progression of heart failure. Curcumin (CUR), a natural p300-specific HAT inhibitor, suppresses hypertrophic responses and prevents deterioration of left-ventricular systolic function in heart-failure models. However, few structure-activity relationship studies on cardiomyocyte hypertrophy using CUR have been conducted. To evaluate if prenylated pyrazolo curcumin (PPC) and curcumin pyrazole (PyrC) can suppress cardiomyocyte hypertrophy, cultured cardiomyocytes were treated with CUR, PPC, or PyrC and then stimulated with phenylephrine (PE). PE-induced cardiomyocyte hypertrophy was inhibited by PyrC but not PPC at a lower concentration than CUR. Western blotting showed that PyrC suppressed PE-induced histone acetylation. However, an in vitro HAT assay showed that PyrC did not directly inhibit p300-HAT activity. As Cdk9 phosphorylates both RNA polymerase II and p300 and increases p300-HAT activity, the effects of CUR and PyrC on the kinase activity of Cdk9 were examined. Phosphorylation of p300 by Cdk9 was suppressed by PyrC. Immunoprecipitation-WB showed that PyrC inhibits Cdk9 binding to CyclinT1 in cultured cardiomyocytes. PyrC may prevent cardiomyocyte hypertrophic responses by indirectly suppressing both p300-HAT activity and RNA polymerase II transcription elongation activity via inhibition of Cdk9 kinase activity.

Pitfalls in the structural elucidation of small molecules. A critical analysis of a decade of structural misassignments of marine natural products.

Covering: July 2010 to August 2021This article summarizes more than 200 cases of misassigned marine natural products reported between July 2010 and August 2021, sorting out errors according to the structural elements. Based on a comparative analysis of the original and the revised structures, major pitfalls still plaguing the structural elucidation of small molecules were identified, emphasizing the role of total synthesis, crystallography, as well as chemical- and biosynthetic logic to complement spectroscopic data. Distinct "trends" in natural product misassignment are evident between compounds of marine and plant origin, with an overall much lower incidence of "impossible" structures within misassigned marine natural products.

A Nrf-2 Stimulatory Hydroxylated Cannabidiol Derivative from Hemp (Cannabis sativa).

A phytochemical analysis of mother liquors obtained from crystallization of CBD from hemp (Cannabis sativa), guided by LC-MS/MS and molecular networking profiling and completed by isolation and NMR-based characterization of constituents, resulted in the identification of 13 phytocannabinoids. Among them, anhydrocannabimovone (5), isolated for the first time as a natural product, and three new hydroxylated CBD analogues (1,2-dihydroxycannabidiol, 6, 3,4-dehydro-1,2-dihydroxycannabidiol, 7, and hexocannabitriol, 8) were obtained. Hexocannabitriol (8) potently modulated, in a ROS-independent way, the Nrf2 pathway, outperforming all other cannabinoids obtained in this study and qualifying as a potential new chemopreventive chemotype against cancer and other degenerative diseases.

Shedding light on curcumin stability.

The stability of molecular curcumin (purcumin, 1a) in solution is strongly light-dependent. Under laboratory artificial light, a relative stability is observed only at neutral pH, while more intense light and/or solar light can trigger degradation via a combination of hydrolytic and oxidative fragmentation of the heptadiendione moiety. Minor curcuminoids in commercial curcumin (purcuminoids) can improve the stability of molecular curcumin, but only under conditions of low irradiation. While confirming earlier observations alerting to the instability of purcumin, our results provide new rationales for unexplained differences between previous studies, question the biological relevance of a non-enzymatic degradation for the bioactivity profiles that have been reported for purcumin, and highlight the need of a better characterization of the degradation of purcuminoids under visible light irradiation.

Agathadiol, a labdane diterpenoid from juniper berries, is a positive allosteric modulator of CB1R.

The neutral fraction of a juniper (Juniperus communis L.) berries acetone extract could positively modulate the activity of type 1 - cannabinoid receptor (CB1R). Bioactivity-directed fractionation identified the labdane diterpenoid agathadiol (4) as a positive allosteric modulator of CB1R, while closely related analogues were inactive. Agathadiol (4) is a minor constituent of juniper, but could be more conveniently obtained by semisynthesis from agathic acid (8), a major constituent of Manila copal.

Cannabinoquinones: Synthesis and Biological Profile.

Neutral cannabinoids are oxidatively unstable and are converted into quinone derivatives by atmospheric- and/or chemical oxidative dearomatization. The study of cannabinoquinones has long been plagued by their lability toward additional oxidative degradation, but full substitution of the quinone ring, as well as the introduction of steric hindrance on the alkyl substituent, have provided sufficient stability for a systematic investigation of their bioactivity and for further clinical development. These studies culminated in the discovery of the aminocannabinoquinone VCE-004.8 (5), a compound under phase 2 clinical development with orphan drug status by EMA and FDA for the management of scleroderma. The synthesis and rich chemistry of these compounds will be described, summarizing their biological profile and clinical potential.

The Combined Effect of Branching and Elongation on the Bioactivity Profile of Phytocannabinoids. Part I: Thermo-TRPs.

The affinity of cannabinoids for their CB1 and CB2 metabotropic receptors is dramatically affected by a combination of α-branching and elongation of their alkyl substituent, a maneuver exemplified by the n-pentyl -> α,α-dimethylheptyl (DMH) swap. The effect of this change on other cannabinoid end-points is still unknown, an observation surprising since thermo-TRPs are targeted by phytocannabinoids with often sub-micromolar affinity. To fill this gap, the α,α-dimethylheptyl analogues of the five major phytocannabinoids [CBD (1a), Δ8-THC (6a), CBG (7a), CBC (8a) and CBN (9a)] were prepared by total synthesis, and their activity on thermo-TRPs (TRPV1-4, TRPM8, and TRPA1) was compared with that of one of their natural analogues. Surprisingly, the DMH chain promoted a shift in the selectivity toward TRPA1, a target involved in pain and inflammatory diseases, in all investigated compounds. A comparative study of the putative binding modes at TRPA1 between DMH-CBC (8b), the most active compound within the series, and CBC (8a) was carried out by molecular docking, allowing the rationalization of their activity in terms of structure-activity relationships. Taken together, these observations qualify DMH-CBC (8b) as a non-covalent TRPA1-selective cannabinoid lead that is worthy of additional investigation as an analgesic and anti-inflammatory agent.