Exploring quercetin and luteolin derivatives as antiangiogenic agents

The formation of new blood vessels from the pre-existing vasculature (angiogenesis) is a crucial stage in cancer progression and, indeed, angiogenesis inhibitors are now used as anticancer agents, clinically. Here we have explored the potential of flavonoid derivatives as antiangiogenic agents. Specifically, we have synthesised methoxy and 4-thio derivatives of the natural flavones quercetin and luteolin, two of which (4-thio quercetin and 4-thio luteolin) had never been previously reported. Seven of these compounds showed significant (P<0.05) antiangiogenic activity in an in vitro scratch assay. Their activity ranged from an 86% inhibition of the vascular endothelium growth factor (VEGF)-stimulated migration (observed for methoxyquercetin at 10 µM and for luteolin at 1 µM) to a 36% inhibition (for thiomethoxy quercetin at 10 µM). Western blotting studies showed that most (4 out of 7) compounds inhibited phosphorylation of the VEGF receptor-2 (VEGFR2), suggesting that the antiangiogenic activity was due to an interference with the VEGF/VEGFR2 pathway. Molecular modelling studies looking at the affinity of our compounds towards VEGFR and/or VEGF confirmed this hypothesis, and indeed the compound with the highest antiangiogenic activity (methoxyquercetin) showed the highest affinity towards VEGFR and VEGF. As reports from others have suggested that structurally similar compounds can elicit biological responses via a non-specific, promiscuous membrane perturbation, potential interactions of the active compounds with a model lipid bilayer were assessed via DSC. Luteolin and its derivatives did not perturb the model membrane even at concentrations 10 times higher than the biologically active concentration and only subtle interactions were observed for quercetin and its derivatives. Finally, cytotoxicity assessment of these flavonoid derivatives against MCF-7 breast cancer cells demonstrated also a direct anticancer activity albeit at generally higher concentrations than those required for an antiangiogenic effect (10 fold higher for the methoxy analogues). Taken together these results show promise for flavonoid derivatives as antiangiogenic agents

Recent Advances and Impact of Chemotherapeutic and Antiangiogenic Nanoformulations for Combination Cancer Therapy

Traditional chemotherapy, along with antiangiogenesis drugs (combination cancer therapy), has shown reduced tumor recurrence and improved antitumor effects, as tumor growth and metastasis are often dependent on tumor vascularization. However, the effect of combination chemotherapy, including synergism and additive and even antagonism effects, depends on drug combinations in an optimized ratio. Hence, nanoformulations are ideal, demonstrating a great potential for the combination therapy of chemo-antiangiogenesis for cancer. The rationale for designing various nanocarriers for combination therapy is derived from organic (polymer, lipid), inorganic, or hybrid materials. In particular, hybrid nanocarriers that consist of more than one material construct provide flexibility for different modes of entrapment within the same carrier-e.g., physical adsorption, encapsulation, and chemical conjugation strategies. These multifunctional nanocarriers can thus be used to co-deliver chemo- and antiangiogenesis drugs with tunable drug release at target sites. Hence, this review attempts to survey the most recent advances in nanoformulations and their impact on cancer treatment in a combined regimen-i.e., conventional cytotoxic and antiangiogenesis agents. The mechanisms and site-specific co-delivery strategies are also discussed herein, along with future prospects

Thioflavones as novel neuroprotective agents

Oxidative stress is associated with the pathology of neurodegenerative diseases. Identification of small molecules capable of protecting against oxidative stress is therefore of significant importance. In this context, a library of 76 hydroxy flavones, methoxy flavones and their 4-thio analogues has been evaluated for neuroprotection against H2O2-induced oxidative stress. This revealed the synthetic 7,8-dihydroxy 4-thioflavones as neuroprotective compounds, with 14d and 18d showing highest neuroprotective effects at lower concentrations (0.3 μM). Neuroprotection was found to be mediated via activation of the anti-apoptotic cell survival proteins of the ERK1/2 and PI3K/Akt pathways. Structure-activity relationship analysis revealed the B-ring phenyl group as essential for greater neuroprotection. Replacing the 4-C=O moiety with a 4-C=S moiety also generally enhanced neuroprotection

Synthetic flavonoids as novel modulators of platelet function and thrombosis

Cardiovascular diseases represent a major cause of mortality and morbidity in the world and thrombotic conditions such as heart attacks and strokes are caused by unwarranted activation of platelets and subsequent formation of blood clots (thrombi) within the blood vessels during pathological circumstances. Therefore, platelets act as a primary therapeutic target to treat and prevent thrombotic conditions. Current treatments are limited due to intolerance and they are associated with severe side effects such as bleeding complications. Hence, the development of novel therapeutic strategies for thrombotic diseases is an urgent priority. Flavonoids are naturally occurring plant-derived molecules that exert numerous beneficial effects in humans through modulating the functions of distinct cell types. However, naturally occurring flavonoids suffer from several issues such as poor solubility, lipophilicity, and bioavailability, which hinder their efficacy and potency. Despite this, flavonoids act as versatile templates for the design and synthesis of novel molecules for various therapeutic targets. Indeed, several synthetic flavonoids have recently been developed to improve their stability, bioavailability and efficacy including for the modulation of platelet function. Here, we provide insight into the actions of certain natural flavonoids along with the advantages of synthetic flavonoids in the modulation of platelet function, haemostasis and thrombosis

Repurposing cancer drugs, batimastat and marimastat, to inhibit the activity of a group I metalloprotease from the venom of the Western Diamondback rattlesnake, Crotalus atrox

Snakebite envenomation causes over 140,000 deaths every year predominantly in developing countries. As a result, it is one of the most lethal neglected tropical diseases. It is associated with an incredibly complex pathophysiology due to the vast number of unique toxins/proteins found in the venoms of diverse snake species found worldwide. Here, we report the purification and functional characteristics of a group I metalloprotease (CAMP-2) from the venom of the western diamondback rattlesnake, Crotalus atrox. Its sensitivity to matrix metalloprotease inhibitors (batimastat and marimastat) was established using specific in vitro experiments and in silico molecular docking analysis. CAMP-2 shows high sequence homology to atroxase from the venom of Crotalus atrox and exhibits collagenolytic, fibrinogenolytic and mild haemolytic activities. It exerts a mild inhibitory effect on agonist-induced platelet aggregation in the absence of plasma proteins. Its collagenolytic activity was completely inhibited by batimastat and marimastat. Zinc chloride also inhibits the collagenolytic activity of CAMP-2 by around 75% at 50 M, while it is partially potentiated by calcium chloride. Molecular docking studies demonstrate that batimastat and marimastat are able to bind strongly to the active site residues of CAMP-2. This study demonstrates the impact of matrix metalloprotease inhibitors in the modulation of a purified, group I metalloprotease activities in comparison to the whole venom. By improving our understanding of snake venom metalloproteases and their sensitivity to small molecule inhibitors, we can begin to develop novel and improved treatment strategies for snakebites

The antimicrobial cathelicidin CRAMP augments platelet activation during psoriasis in mice

Platelet-associated complications including thrombosis, thrombocytopenia, and haemorrhage are commonly observed during various inflammatory diseases such as psoriasis. Although several mechanisms that may contribute to the dysfunction of platelets during inflammatory diseases have been reported, knowledge on the primary molecules/mechanisms that underpin platelet-associated complications in such conditions is not fully established. Here, we report the significance of the mouse antimicrobial cathelicidin, mouse cathelicidin-related antimicrobial peptide (mCRAMP) (an orthologue of LL37 in humans), on the modulation of platelet reactivity during psoriasis using Imiquimod-induced psoriasis in mice as an inflammatory disease model for psoriasis vulgaris in humans. The activation of platelets during psoriasis is increased as evidenced by the elevated levels of fibrinogen binding and P-selectin exposure on the surface of platelets, and the level of soluble P-selectin in the plasma of psoriatic mice. The skin and plasma of psoriatic mice displayed increased levels of mCRAMP. Moreover, the plasma of psoriatic mice augmented the activation of platelets obtained from healthy mice. The effect of mCRAMP is partially mediated through formyl peptide receptor 2/3 (Fpr2/3, the orthologue to human FPR2/ALX) in platelets as a significant reduction in their activation was observed when FPR2/ALX-selective inhibitors such as WRW4 or Fpr2/3-deficient mouse platelets were used in these assays. Since the level of antimicrobial cathelicidin is increased in numerous inflammatory diseases such as psoriasis, atherosclerosis, and inflammatory bowel disease, the results of this study point towards a critical role for antimicrobial cathelicidin and FPR2/ALX in the development of platelet-related complications in such diseases

1,8-cineole affects agonists-induced platelet activation, thrombus formation and haemostasis

1,8-cineole, a monoterpenoid is a major component of eucalyptus oil and has been proven to possess numerous beneficial effects in humans. Notably, 1,8-cineole is the primary active ingredient of a clinically approved drug, Soledum® which is being mainly used for the maintenance of sinus and respiratory health. Due to its clinically valuable properties, 1,8-cineole has gained significant scientific interest over the recent years specifically to investigate its anti-inflammatory and antioxidant effects. However, the impact of 1,8-cineole on the modulation of platelet activation, thrombosis and haemostasis was not fully established. Therefore, in this study, we demonstrate the effects of 1,8-cineole on agonists-induced platelet activation, thrombus formation under arterial flow conditions and haemostasis in mice. 1,8-cineole largely inhibits platelet activation stimulated by glycoprotein VI (GPVI) agonists such as collagen and cross-linked collagen-related peptide (CRP-XL), while it displays minimal inhibitory effects on thrombin or ADP-induced platelet aggregation. It inhibited inside-out signalling to integrin αIIbβ3 and outside-in signalling triggered by the same integrin as well as granule secretion and intracellular calcium mobilisation in platelets. 1,8-cineole affected thrombus formation on collagen-coated surface under arterial flow conditions and displayed a minimal effect on haemostasis of mice at a lower concentration of 6.25 µM. Notably, 1,8-cineole was found to be non-toxic to platelets up to 50 µM concentration. The investigation on the molecular mechanisms through which 1,8-cineole inhibits platelet function suggests that this compound affects signalling mediated by various molecules such as AKT, Syk, LAT, and cAMP in platelets. Based on these results, we conclude that 1,8-cineole may act as a potential therapeutic agent to control unwarranted platelet reactivity under various pathophysiological settings

Metal complexes of flavonoids: their synthesis, characterization, and enhanced anti-oxidant and anti-cancer activities

Flavonoids are polyphenolic compounds of natural origin. They are extensively studied within drug discovery programmes due to their wide ranging biological activities such as anti-microbial, anti-oxidant, anti-tumor, neuroprotective and cardioprotective properties. The ability of flavonoids to coordinate with metal atoms has provided new leads for drug discovery programmes, with better pharmacological activities and clinical profiles than the parent flavonoids. In this review, the enhanced anti-oxidant and anti-cancer activities of flavonoid metal complexes versus the parent flavonoids are discussed. Possible mechanisms of action for the metal complexes, such as DNA binding and apoptosis induction, are also presented alongside an overview of the synthesis of the metal complexes, and the different techniques used for their characterization

The endogenous antimicrobial cathelicidin LL37 induces platelet activation and augments thrombus formation

Platelet-associated complications including thrombosis, thrombocytopenia and haemorrhage are commonly observed during various inflammatory diseases such as psoriasis, sepsis and inflammatory bowel disease. Despite the reported evidence on numerous mechanisms/molecules that may contribute to the dysfunction of platelets, the primary mechanisms that underpin platelet-associated complications during inflammatory diseases are not fully established. Here, we report the discovery of formyl peptide receptor 2, FPR2/ALX in platelets, and its primary role in the development of platelet-associated complications via ligation with its ligand, LL37. LL37 acts as a powerful endogenous antimicrobial peptide but it also regulates innate immune responses. We demonstrate the impact of LL37 in the modulation of platelet reactivity, haemostasis, and thrombosis. LL37 activates a range of platelet functions, enhances thrombus formation, and shortens the tail bleeding time in mice. By utilising a pharmacological inhibitor and Fpr2/3 (an orthologue of human FPR2/ALX)-deficient mice, the functional dependence of LL37 on FPR2/ALX was determined. Since the level of LL37 is increased in numerous inflammatory diseases, these results point towards a critical role for LL37 and FPR2/ALX in the development of platelet-related complications in such diseases. Hence, a better understanding of the clinical relevance of LL37 and FPR2/ALX in diverse pathophysiological settings will pave the way for the development of improved therapeutic strategies for a range of thromboinflammatory diseases

Impact of Naja nigricollis Venom on the Production of Methaemoglobin

Snakebite envenomation is an affliction currently estimated to be killing upwards of 100,000 people annually. Snakebite is associated with a diverse pathophysiology due to the magnitude of variation in venom composition that is observed worldwide. The haemolytic (i.e., lysis of red blood cells) actions of snake venoms are well documented, although the direct impact of venoms on haemoglobin is not fully understood. Here we report on the varied ability of a multitude of snake venoms to oxidise haemoglobin into methaemoglobin. Moreover, our results demonstrate that the venom of an elapid, the black necked spitting cobra, Naja nigricollis, oxidises oxyhaemoglobin (Fe2+) into methaemoglobin (Fe3+) in a time- and concentration-dependent manner that is unparalleled within the 47 viper and elapid venoms evaluated. The treatment of venom with a reducing agent, dithiothreitol (DTT) is observed to potentiate this effect at higher concentrations, and the use of denatured venom demonstrates that this effect is dependent upon the heat-sensitive proteinaceous elements of the venom. Together, our results suggest that Naja nigricollis venom appears to promote methaemoglobin production to a degree that is rare within the Elapidae family, and this activity appears to be independent of proteolytic activities of venom components on haemoglobin