Synthesis and anxiolytic effect of europium metallic complex containing lapachol [Eu(DBM)3. LAP] in adult zebrafish through serotonergic neurotransmission: in vivo and in silico approach.

Anxiety-related mental health problems are estimated at 3.6% globally, benzodiazepines (BZDs) are the class of drugs indicated for the treatment of anxiety, including lorazepam and diazepam. However, concerns have been raised about the short- and long-term risks associated with BZDs. Therefore, despite anxiolytic and antidepressant drugs, there is a need to develop more effective pharmacotherapies with fewer side effects than existing drugs. The present work reported the synthesis, anxiolytic activity, mechanism of action in Adult Zebrafish (Danio rerio) and in silico study of a europium metallic complex with Lapachol, [Eu(DBM)3. LAP]. Each animal (n = 6/group) was treated intraperitoneally (i.p.; 20 µL) with the synthesized complex (4, 20 and 40 mg/Kg) and with the vehicle (DMSO 3%; 20 µL), being submitted to the tests of locomotor activity and 96h acute toxicity. The light/dark test was also performed, and the serotonergic mechanism (5-HT) was evaluated through the antagonists of the 5-HTR1, 5-HTR2A/2C and 5-HTR3A/3B receptors. The complex was characterized using spectrometric techniques, and the anxiolytic effect of complex may be involved the neuromodulation of receptors 5-HT3A/3B, since the pre-treatment with pizotifen and cyproheptadine did not block the anxiolytic effect of [Eu(DBM)3. LAP], unlike fluoxetine had its anxiolytic effect reversed. In addition, molecular docking showed interaction between the [Eu(DBM)3. LAP] and 5HT3A receptor with binding energy -7.8 kcal/mol and the ADMET study showed that complex has low toxic risk. It is expected that the beginning of this study will allow the application of the new anxiolytic drugs, given the pharmacological potential of the lapachol complex.Communicated by Ramaswamy H. Sarma.

Preclinical evaluation of L-fucoside from lapachol-loaded nanoemulsion as a strategy to breast cancer treatment.

Breast cancer prevails as the most common cancer in women, underscoring an urgent need for more effective therapies. This study explores the potential of our newly developed nanoemulsion containing a novel fucoside derivative of lapachol (NE-F-LapA) as an intravenous treatment strategy. We sought to overcome the solubility issues associated with fucoside with this improved drug delivery strategy that enhances tumor delivery and mitigates other dose-limiting toxicities. Nanoemulsion was prepared and characterized by DLS, zeta potential, encapsulation efficiency, and storage stability. Cytotoxicity against breast cancer cell lines (4T1 and MDA-MB-231) and non-tumor human fibroblasts (NTHF) were evaluated. In vivo assays included antitumoral activity performance and acute systemic toxicity in mice models. NE-F-LapA was synthesized and optimized to 200 nm size, - 20 mV zeta potential, and near-complete (>98%) drug encapsulation. Stability exceeded 6 months, and biological fluid exposure maintained suitable properties for administration. In vitro, NE-F-LapA showed high toxicity (3 µM) against 4T1 and MDA-MB-231, enhanced five times the breast cancer cell uptake and three times the selectivity when compared to normal cells. Systemic toxicity assessment in mice revealed no concerning hematological or biochemical changes. Finally, in a 4T1 breast tumor model, NE-F-LapA significantly inhibited growth by 50% of the subcutaneous 4T1 tumor and reduced lung metastases 5-fold versus control. Overall, tailored nanoemulsification of the lapachol derivative enabled effective intravenous administration and improved efficacy over the free drug, indicating promise for enhanced breast cancer therapy pending further optimization.

Density functional theory, molecular docking, In vitro and In vivo anti-inflammatory investigation of lapachol isolated from Fernandoaadenophylla.

Medicinal plants are the main source of active chemical constituents responsible for curing or mitigating various ailments. To discover new, safe, and effective drug candidates the isolation and screening of natural products are essential. In the current research work, lapachol was isolated from Fernandoa adenophylla, which was evaluated for anti-inflammatory effect followed by molecular docking. The isolated compound was tested for anti-inflammatory effects using in vitro (HRBC assay) and in vivo (xylene-induced ear edema) experimental models. Various concentrations of lapachol demonstrated anti-inflammatory effects with a percent potential of 77.96 at 100 µM. Different concentrations of Lapachol demonstrated a dose-dependent anti-edematous effect with a maximum percent effect of 77.9 % at a higher dose. The histopathological study revealed that the application of xylene led to a significant increase in ear thickness, along with clear signs of ear edema and infiltration of inflammatory cells, as well as epidermal hyperplasia of the dermis when compared to the control group. However, treatment with the investigated compound showed a significant reduction in ear thickness and pathological differences comparable to those observed in the group treated with diclofenac. Density functional theory calculations are accomplished to gain insight into structural and spectroscopic properties. Geometry optimization, FMO, and MEP analyses are performed. Overall, the molecular docking results indicate that lapachol has potential as a COX inhibitor by binding to the active sites of both COX-1 and COX-2 enzymes.

Identification of highly active compounds from insecticidal plant Oroxylum indicum L. (Vent.) and the induction of apoptosis by lapachol on Sf9 cells.

The extraction of biopesticides from plants has become a promising field for agricultural development. To explore a high-efficiency and viable method for the screening of plant compounds with insecticidal activity, we screened for active ingredients in the insecticidal plant, Oroxylum indicum L. Vent, using Sf9 cells. A CCK-8 cytotoxicity assay kit was used for high-throughput screening of 34 compounds contained in O. indicum. The apoptosis-inducing effect of the highly cytotoxic compound on Sf9 cells was investigated by morphological characterization using inverted microscopy, caspase-3 activity assay, and DNA gel electrophoresis. Finally, the biological activity of compounds against aphids was evaluated using the leaf-pest dipping methods and leaf dipping methods. Results showed that among the main compounds identified, lapachol, chrysin, and baicalein had good proliferation inhibitory effects on Sf9 cells, with their recorded IC50 being 11.53 mg/L, 38.39 mg/L, and 42.10 mg/L, respectively. Moreover, the IC50 value of lapachol was lower than the control insecticides rotenone (18.03 mg/L) and fipronil (21.04 mg/L). Apoptosis assay further showed that lapachol promoted the production of caspase-3 and led to DNA fragmentation in Sf9 cells. Lapachol showed high biological activity against Aphis gossypii, Sitobion avenae, and Semiaphis heraclei, with its recorded LC50 being 104.40, 101.80, and 110.29 mg/L, respectively, which were comparable to the activity of the control insecticide rotenone. High-throughput screening of active ingredients in the insecticidal plant O. indicum using Sf9 cells is feasible, and the identification of lapachol as the main aphidicidal active substance is valuable for further study.

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

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

Lapachol treats non-alcoholic fatty liver disease by modulating the M1 polarization of Kupffer cells via PKM2.

AIM: This study investigated the mechanism of action of lapachol (LAP) against non-alcoholic fatty liver disease (NAFLD). METHODS: Primary Kupffer cells (KCs) of rats were used for in-vitro experiments. The proportion of M1 cells was assayed by flow cytometry, the levels of M1 inflammatory markers were determined by enzyme-linked immunosorbent assay (ELISA) combined with real-time quantitative fluorescence PCR (RT-qPCR), the expression of p-PKM2 was detected by Western-Blotting. A SD rat model of NAFLD was established with high-fat diet. Following LAP intervention, the changes in blood glucose/lipid, insulin resistance and liver function were detected, and the hepatic histopathologic changes were examined by histological staining. RESULTS: The results showed that LAP could inhibit the M1 polarization of KCs, lower the levels of inflammatory cytokines, and suppress the activation of PKM2. The effect of LAP could be counteracted after using PKM2 inhibitor PKM2-IN-1 or knocking out PKM2. Small molecule docking revealed that LAP could inhibit the phosphorylation process of PKM2 by binding to ARG-246, the phosphorylation site of PKM2. In rat experiments, LAP could ameliorate the liver function and lipid metabolism of NAFLD rats, and inhibit the hepatic histopathologic changes. CONCLUSION: Our study found that LAP can inhibit the phosphorylation of PKM2 by binding to PKM2-ARG-246, thereby regulating the M1 polarization of KCs and inhibiting the inflammatory response of liver tissues to treat NAFLD. LAP has potential as a novel pharmaceutical for treating NAFLD.

Novel Ru(II)-bipyridine/phenanthroline-lapachol complexes as potential anti-cancer agents.

For the first time, we herein report on the syntheses of two new Ru(II)/bipyridine/phenanthroline complexes containing lapachol as ligand: complex (1), [Ru (bipy)2(Lap)]PF6 and complex (2), [Ru(Lap)(phen)2]PF6, where bipy = 2,2'-bipyridine and ph en = 1,10-phenanthroline; Lap = lapachol (2-hydroxy-3-(3-methylbut-2-en-1- yl)naphthalene-1,4-dione). The complexes were synthesized and characterized by elemental analyses, molar conductivity, mass spectrometry, ultraviolet-visible and infrared spectroscopies, nuclear magnetic resonance (1H, 13C), and single crystal X-ray diffraction, for complex (2). In addition, in vitro cytotoxicity was tested against six cancer cells: A549 (lung carcinoma); DU-145 (human prostate carcinoma); HepG2 (human hepatocellular carcinoma), PC-3 (human prostate adenocarcinoma); MDA-MB-231 (human breast adenocarcinoma); Caco-2 (human colorectal adenocarcinoma), and against two non-cancer cells, FGH (human gingival normal fibroblasts) and PNT-2 (prostate epithelial cells). Complex (1) was slightly more toxic and selective than complex (2) for all cell lines, except against the A549 cells, where (2) was more potent than complex (1). The complexes induced an increase in the reactive oxygen species, and the co-treatment with N-acetyl-L-cysteine remarkably suppressed the ROS generation and prevented the reduction of cell viability, suggesting that the cytotoxicity of the complexes is related to the ROS-mediated pathway. Further studies indicated that the complexes may bind to DNA via minor groove interaction. Our studies also revealed that free Lap induces gene mutations in Salmonella Typhimurium, nevertheless, the complexes demonstrated the absence of genotoxicity by the Ames test. The present study provides a relevant contribution to understanding the anti-cancer potential and genetic toxicological events of new ruthenium complexes containing the lapachol molecule as a ligand.

Naphthoquinones and derivatives as potential anticancer agents: An updated review.

One of the leading global causes of death is cancer; even though several treatment methods have improved survival rates, the incidence and fatality rates remain high. Naphthoquinones are a type of quinone that is found in nature and has vital biological roles. These chemicals have anticancer (antineoplastic), analgesic, anti-inflammatory, antimalarial, antifungal, antiviral, antitrypanosomal, antischistosomal, leishmanicidal, and anti-ulcerative effects. Direct addition of a substituent group to the 1,4-naphthoquinone ring can alter the naphthoquinone's oxidation/reduction and acid/base characteristics, and the activity can be altered. Because of their pharmacological properties, such as anticancer activity and probable therapeutic application, naphthoquinones have greatly interested the scientific community. Some chemicals having a quinone ring in malignant cells have been found to have antiproliferative effects. Naphthoquinones' deadly impact is connected with the inhibition of electron transporters, the uncoupling of oxidative phosphorylation, the creation of ROS, and the formation of protein adducts, notably with -SH enzyme groups. This review article aims to discuss naphthoquinones and their derivatives, which act against cancer and their future perspectives. This review covers several studies highlighting the potent anticancer properties of naphthoquinones. Further, various proposed mechanisms of anticancer actions of naphthoquinones have been summarized in this review.

In silico evaluation of lapachol derivatives binding to the Nsp9 of SARS-CoV-2.

SARS-CoV-2 is the etiological agent of COVID-19, which represents a global health emergency that was rapidly declared a pandemic by the World Health Organization. Currently, there is a dearth of effective targeted therapies against viruses. Natural products isolated from traditional herbal plants have had a huge impact on drug development aimed at various diseases. Lapachol is a 1,4- naphthoquinone compound that has been demonstrated to have therapeutic effects against several diseases. SARS-CoV-2 non-structural proteins (nsps) play an important role in the viral replication cycle. Nsp9 seems to play a key role in transcription of the RNA genome of SARS-CoV-2. Virtual screening by docking and molecular dynamics suggests that lapachol derivatives can interact with Nsp9 from SARS-CoV-2. Complexes of lapachol derivatives V, VI, VIII, IX, and XI with the Nsp9 RNA binding site were subjected to molecular dynamics assays, to assess the stability of the complexes via RMSD. All complexes were stable over the course of 100 ns dynamics assays. Analyses of the hydrogen bonds in the complexes showed that lapachol derivatives VI and IX demonstrated strongest binding, with a stable or increasing number of hydrogen bonds over time. Our results demonstrate that Nsp9 from SARS-CoV-2 could be an important target in prospecting for ligands with antiviral potential. In addition, we showed that lapachol derivatives are potential ligands for SARS-CoV-2 Nsp9.Communicated by Ramaswamy H. Sarma.

Chemoselective Synthesis of Mannich Adducts from 1,4-Naphthoquinones and Profile as Autophagic Inducers in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a worldwide public health problem, accounting for approximately 90% of all oral cancers, and is the eighth most common cancer in men. Cisplatin and carboplatin are the main chemotherapy drugs used in the clinic. However, in addition to their serious side effects, such as damage to the nervous system and kidneys, there is also drug resistance. Thus, the development of new drugs becomes of great importance. Naphthoquinones have been described with antitumor activity. Some of them are found in nature, but semi synthesis has been used as strategy to find new chemical entities for the treatment of cancer. In the present study, we promote a multiple component reaction (MCR) among lawsone, arylaldehydes, and benzylamine to produce sixteen chemoselectively derivated Mannich adducts of 1,4-naphthoquinones in good yield (up to 97%). The antitumor activities and molecular mechanisms of action of these compounds were investigated in OSCC models and the compound 6a induced cytotoxicity in three different tumor cell lines (OSCC4, OSCC9, and OSCC25) and was more selective (IS > 2) for tumor cells than the chemotropic drug carboplatin and the controls lapachol and shikonin, which are chemically similar compounds with cytotoxic effects. The 6a selectively and significantly reduced the amount of cell colony growth, was not hemolytic, and tolerable in mice with no serious side effects at a concentration of 100 mg/kg with a LD50 of 150 mg/kg. The new compound is biologically stable with a profile similar to carboplatin. Morphologically, 6a does not induce cell retraction or membrane blebs, but it does induce intense vesicle formation and late emergence of membrane bubbles. Exploring the mechanism of cell death induction, compound 6a does not induce ROS formation, and cell viability was not affected by inhibitors of apoptosis (ZVAD) and necroptosis (necrostatin 1). Autophagy followed by a late apoptosis process appears to be the death-inducing pathway of 6a, as observed by increased viability by the autophagy inhibitor (3-MA) and by the appearance of autophagosomes, later triggering a process of late apoptosis with the presence of caspase 3/7 and DNA fragmentation. Molecular modeling suggests the ability of the compound to bind to topoisomerase I and II and with greater affinity to hPKM2 enzyme than controls, which could explain the mechanism of cell death by autophagy. Finally, the in-silico prediction of drug-relevant properties showed that compound 6a has a good pharmacokinetic profile when compared to carboplatin and doxorubicin. Among the sixteen naphthoquinones tested, compound 6a was the most effective and is highly selective and well tolerated in animals. The induction of cell death in OSCC through autophagy followed by late apoptosis possibly via inhibition of the PKM2 enzyme points to a promising potential of 6a as a new preclinical anticancer candidate.

Lapachol-Induced Upregulation of Sirt1/Sirt3 is linked with Improved Skin Wound Healing in Alloxan-induced Diabetic Mice.

Timely repair of damaged skin is very important to maintain the integrity and homeostasis of skin, but the wound healing process is compromised in diabetic patients due to several extrinsic and intrinsic factors thus lead to leg amputation and death eventually. Sirtuins, a family of seven conserved proteins are known to be associated with pathophysiological processes of the skin. The most important among them are sirt1and sirt3 involved in cell regeneration and cell survival. Naphthoquinone derivatives have a wide range of therapeutic properties, but the potential diabetic wound healing activity of lapachol has not been identified yet. The present study thus aimed to investigate the wound healing effects of lapachol in a diabetic mouse model. Diabetic wounded mice were divided into 3 groups; vehicle, lapachol 0.05%, and lapachol 0.1%. Skin samples collected from diabetic wounded mice on different time points after treatment for 10 consecutive days were subjected to downstream analysis by western blot, ELISA and histology. Lapachol treatment was found to enhance the expression of sirt1/sirt3 and other proteins involved in cell migration and blood vessel formation. The tissue development rate was increased by lapachol treatment with better collagen deposition. Interestingly, lapachol treatment also gave rise to a high concentration of growth factors resulting in speedy and timely recovery of injured skin. In summary, our findings suggest that lapachol promotes efficient wound healing in a diabetic mouse model by increasing the expression of sirt1 and sirt3 and other proteins related to wound repair and skin regeneration including α-PAK, RAC1/CDC42, VEGF and growth factors viz PDGF and VEGF. This research work finds a novel potential activator of sirtuins in the form of lapachol and depicts the role of activated sirtuins in diabetic wound healing.

A Novel Ruthenium(II) Complex With Lapachol Induces G2/M Phase Arrest Through Aurora-B Kinase Down-Regulation and ROS-Mediated Apoptosis in Human Prostate Adenocarcinoma Cells.

Lapachol is a well-studied natural product that has been receiving great interest due to its anticancer properties that target oxidative stress. In the present work, two novel lapachol-containing ruthenium(II) complexes [Ru(Lap)(dppm)(bipy)]PF6 (1) and [Ru(Lap)(dppm)(phen)]PF6 (2) [Lap = lapachol, dppm = 1,1'-bis(diphosphino)methane, bipy = 2,2'-bipyridine, phen = 1,10-phenantroline] were synthesized, fully characterized, and investigated for their cellular and molecular responses on cancer cell lines. We found that both complexes exhibited a potent cytotoxic effect in a panel of cancer cell lines in monolayer cultures, as well as in a 3D model of multicellular spheroids formed from DU-145 human prostate adenocarcinoma cells. Furthermore, the complex (2) suppressed the colony formation, induced G2/M-phase arrest, and downregulated Aurora-B. The mechanism studies suggest that complex (2) stimulate the overproduction of reactive oxygen species (ROS) and triggers caspase-dependent apoptosis as a result of changes in expression of several genes related to cell proliferation and caspase-3 and -9 activation. Interestingly, we found that N-acetyl-L-cysteine, a ROS scavenger, suppressed the generation of intracellular ROS induced by complex (2), and decreased its cytotoxicity, indicating that ROS-mediated DNA damage leads the DU-145 cells into apoptosis. Overall, we highlighted that coordination of lapachol to phosphinic ruthenium(II) compounds considerably improves the antiproliferative activities of resulting complexes granting attractive selectivity to human prostate adenocarcinoma cells. The DNA damage response to ROS seems to be involved in the induction of caspase-mediated cell death that plays an important role in the complexes' cytotoxicity. Upon further investigations, this novel class of lapachol-containing ruthenium(II) complexes might indicate promising chemotherapeutic agents for prostate cancer therapy.

Anti-melanoma effect of ruthenium(II)-diphosphine complexes containing naphthoquinone ligand.

The use of natural products as potential ligands has been explored as a strategy in the development of metal-based chemotherapy. Since ruthenium complexes are promising alternatives to traditional antitumor agents, this study evaluated the anti-melanoma potential of two ruthenium(II) complexes containing the naphthoquinone ligands lapachol (lap), [Ru(lap)(dppm)2]PF6, and lawsone (law), [Ru(law)(dppm)2]PF6, in addition to the bis(diphenylphosphino)methane (dppm) ligand, referred to as complexes (1) and (2), respectively, using a syngeneic murine melanoma model. Activation of the apoptotic pathway by the treatments was assessed by immunohistochemistry in tumor tissue. Additionally, toxicity of the treatments was evaluated by variation in body and organ weight, quantification of biochemical indicators of renal damage, and genotoxicity in bone marrow and hepatocytes. First, the antiproliferative activity of (1) and (2) was observed in B16F10 cells, with IC50 values of 2.78 and 1.68 µM, respectively. The results obtained in mice showed that, unlike complex (1), (2) possesses significant anti-melanoma activity demonstrated by a reduction in tumor volume and mass (88.42%), as well as in mitosis frequency (83.86%). Additionally, complex (2) increased the levels of cleaved caspase-3, inducing tumor cell apoptosis. When compared to the metallodrug cisplatin, complex (2) exhibited similar anti-melanoma activity and lower toxicity considering all parameters evaluated. In silico studies demonstrated no difference in the binding energy of the naphthoquinone complex between complexes (1) and (2). However, the complex containing the lawsone ligand has a lower molar volume, which may be important for interactions with minor DNA grooves. The present results demonstrate the antitumor efficiency of complex (2) and a significantly lower systemic toxicity compared to cisplatin.

Inhibition of Staphylococcus aureus TetK and MsrA efflux pumps by hydroxyamines derived from lapachol and norlachol.

The present study aimed to evaluate the in vitro efflux pump inhibitory capacity of hydroxyamines derived from lapachol and norlachol, where compounds 3, 4, and 5 were tested against the S. aureus strains: RN4220 carrying the pUL5054 plasmid; and IS-58, endowed with the PT181 plasmid. The substances were synthesized from 2-hydroxy-quinones, lapachol and nor-lapachol obtaining the corresponding 2-methoxylated derivatives via dimethyl sulfate alkylation in a basic medium, which then reacted chemoselectively with 2-ethanolamine and 3-propanolamine to form the corresponding amino alcohols. The antibacterial action of the substances was quantified by determining the Minimum Inhibitory Concentration (MIC), while a microdilution assay was carried out to ascertain efflux pump inhibition of Staphylococcus aureus strains carrying the MsrA macrolide and the TetK tetracycline efflux pumps with the substances at a sub-inhibitory concentration. The results were subjected to statistical analysis by an ANOVA test and Bonferroni post hoc test. The MIC from the substances exhibited a value ≥ 1024 µg/mL. However, a significant reduction (p < 0.0001) of the erythromycin, tetracycline and ethidium bromide MIC was demonstrated when these were in combination with the substances, with this effect being due to a supposed efflux pump inhibition. The tested substances demonstrated effectiveness at decreasing the MIC of erythromycin, tetracycline and ethidium bromide, potentially by inhibiting the MsrA macrolide and the TetK tetracycline efflux pumps present in the tested S. aureus strains.

Enhanced antitumor efficacy of lapachol-loaded nanoemulsion in breast cancer tumor model.

Lapachol (LAP) is a natural compound with various biological properties, including anticancer activity. However, its clinical application is limited due to the low aqueous solubility and potential adverse side effects. Nanoemulsions are drug delivery systems that can assist in the administration of hydrophobic drugs, increasing their bioavailability and protecting from degradation. Thus, this study aimed to prepare a LAP-loaded nanoemulsion (NE-LAP), and evaluate its antitumor activity. For this purpose, the nanoemulsion was prepared using a hot homogenization method and characterized morphologically by cryogenic transmission electron microscopy (cryo-TEM). Mean diameter, polydispersity index, and zeta potential was evaluated by DLS, encapsulation efficiency was measured by HPLC. Moreover, the short-term storage stability, the drug release and hemolysis in vitro was determined. Additionally, pharmacokinetic, toxicology and toxicity properties of99mTc-NE-LAP were evaluated in a breast cancer (4T1) tumor model. The cryo-TEM showed spherical globules, and the physicochemical characterization of NE-LAP showed a homogeneous stable nanoemulsion with a mean diameter of ∼170 nm, zeta potential of around -20 mV, and encapsulation greater than 85 %. In vitro studies validated that encapsulation did not impair the cytotoxicity activity of LAP. The nanoemulsion was successfully radiolabeled and 99mTc-NE-LAP showed prolonged blood circulation and tumor affinity was confirmed by tumor-to-muscle ratio. Moreover, NE-LAP showed higher antitumor activity than the free drug and the treatment did not result in any signs of toxicity. Therefore, these findings suggest that NE-LAP can be considered an effective strategy for cancer treatment.

Effect of Lapachol on the Inhibition of Matrix Metalloproteinase Related to the Invasion of Human Fibrosarcoma Cells.

BACKGROUND: Anti-cancer effect of lapachol contained in Tabebuia avellandae has been poorly understood until now. OBJECTIVE: The aim of this study was to investigate the inhibitory effect of lapachol on MMPs related to cell invasion. Its action mechanism was elucidated by analyzing the activity and the expression of MMPs and the proteins involved in the signaling pathway of cell invasion. METHODS: The cytotoxicity of lapachol was evaluated by MTT assay in HT1080 cells. The effects of lapachol on the expression and the activation of MMPs were analyzed by western blot, immunofluorescence staining, and gelatin zymography assays. Their gene expression was analyzed by RT-PCR, and metastasis was evaluated by cell invasion assay. RESULTS: Lapachol below 2 µM showed no cytotoxicity. It was observed that lapachol above 0.5 µM inhibited the activation of MMP-2 and MMP-9 stimulated by PMA. In particular, the protein and gene expression levels of MMP-2 stimulated by PMA were remarkably decreased in the presence of lapachol at 1 µM compared with the PMA treatment group. In addition, lapachol increased the expression level of TIMP-1 compared with the PMA treatment group. Moreover, lapachol decreased the expression level of p-p38 among MAPKs compared with the PMA treatment group. It was also found that the expression level of p65, a part of NF-kB, in nuclei was reduced in the presence of lapachol above 0.5 µM compared with the PMA treatment group. In addition, lapachol inhibited the invasion of human fibrosarcoma cells stimulated with VEGF. CONCLUSION: Above results suggest that lapachol could play an important role in the modulation of MMPs related to cell invasion via the increase in TIMP-1 expression as well as the inactivation of p38 through NF-kB transcription factor.

Synthesis, trypanocidal and anti-leishmania activity of new triazole-lapachol and nor-lapachol hybrids.

A new library of twenty triazole-lapachol and nor-lapachol derivatives was synthesized. The compounds were evaluated against the epimastigotes form of Trypanosoma cruzi and promastigotes of Leishmania braziliensis and L. infantum. The cytotoxicity of the compounds was determined on murine fibroblasts and used to assess the selectivity index. The introduction of triazole rings in the naphthoquinone derivatives improved activity against the parasitic protozoa T. cruzi and Leishmania species. Some of the derivatives were three to six times more potent than benznidazole against T. cruzi, with similar or slightly better selectivity indexes. The results against L. braziliensis showed that the derivatives 5b and 5e were the most selective compounds. However, they were less selective than the reference compound, miltefosine. Among all products, the derivative 3a was the most selective compound against L. infantum. Nevertheless, it was less potent and less selective than miltefosine. Also, the minimum inhibitory concentration values of the derivatives against nine different bacteria were determined. Moderate antibacterial activity was observed for compound 5c against Staphylococcus aureus.

The plant-derived naphthoquinone lapachol causes an oxidative stress response in Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen, which causes life-threatening systemic and chronic infections and rapidly acquires resistance to multiple antibiotics. Thus, new antimicrobial compounds are required to combat infections with drug resistant S. aureus isolates. The 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone lapachol was previously shown to exert antimicrobial effects. In this study, we investigated the antimicrobial mode of action of lapachol in S. aureus using RNAseq transcriptomics, redox biosensor measurements, S-bacillithiolation assays and phenotype analyses of mutants. In the RNA-seq transcriptome, lapachol caused an oxidative and quinone stress response as well as protein damage as revealed by induction of the PerR, HypR, QsrR, MhqR, CtsR and HrcA regulons. Lapachol treatment further resulted in up-regulation of the SigB and GraRS regulons, which is indicative for cell wall and general stress responses. The redox-cycling mode of action of lapachol was supported by an elevated bacillithiol (BSH) redox potential (EBSH), higher endogenous ROS levels, a faster H2O2 detoxification capacity and increased thiol-oxidation of GapDH and the HypR repressor in vivo. The ROS scavenger N-acetyl cysteine and microaerophilic growth conditions improved the survival of lapachol-treated S. aureus cells. Phenotype analyses revealed an involvement of the catalase KatA and the Brx/BSH/YpdA pathway in protection against lapachol-induced ROS-formation in S. aureus. However, no evidence for irreversible protein alkylation and aggregation was found in lapachol-treated S. aureus cells. Thus, the antimicrobial mode of action of lapachol in S. aureus is mainly caused by ROS formation resulting in an oxidative stress response, an oxidative shift of the EBSH and increased protein thiol-oxidation. As ROS-generating compound, lapachol is an attractive alternative antimicrobial to combat multi-resistant S. aureus isolates.

Molecular mechanism of action of new 1,4-naphthoquinones tethered to 1,2,3-1H-triazoles with cytotoxic and selective effect against oral squamous cell carcinoma.

The oral squamous cell carcinoma (OSCC) stands out as a public health problem due to its high incidence and low survival rate, despite advances in diagnosis and treatment. Moreover, the most commonly chemotherapeutic agents for OSCC, such as carboplatin and cisplatin, generate important side effects, evidencing the urgency in developing new drugs. Naphthoquinones are an important class of natural products or synthetic compounds with cytotoxic effect demonstrated on different cancer types. In the present study, thirty-five 1,4-naphthoquinones tethered to 1,2,3-1H-triazoles were synthesized and the antitumor activity and molecular mechanisms were evaluated in several assays including in vitro and in vivo models of OSCC and normal oral human cells. Compounds 16a, 16b and 16 g were able to induce cytotoxicity in three different tumor cell lines of human OSCC (SCC4, SCC9 and SCC25) and were more toxic and selective to tumor cells (Selective Index, SI > 2) than classical and chemically similar controls (Carboplatin and Lapachol). Compound 16 g showed the higher SI value. Besides, compounds 16a, 16b and 16 g significantly reduced colony formation of SCC9 cells in the tested concentrations. Hemolytic assay using compounds 16a, 16b and 16 g at high concentrations showed no compound exhibited hemolysis higher than 5%, similar to controls. In vivo acute toxicity study showed that 16 g was the only one, among the three compounds, with no apparent limiting toxic effects on mice in the tested concentrations. Thus, the investigation of cell death mechanisms was conducted with this compound. 16 g does not trigger ROS production nor binds to DNA. On the other hand, compound 16 g induced microtubule disorganization, and molecular modeling studies suggests a potential mechanism of action related to inhibition of topoisomerases and/or hPKM2 activities. Cell morphology, pyknotic nuclei presence, cleaved caspase-3 staining and viability assays using caspase-3 inhibitors demonstrate compound 16 g induced cell death through apoptosis. Among the 35 synthesized triazole naphthoquinones, compound 16 g was the most effective compound against OSCC cells, presenting high cytotoxicity (~35 µM), selectivity (SI ~ 6) and low acute toxicity on animals, and therefore might be considered for future cancer therapy.

In silico, in vitro and in vivo evaluation of natural Bignoniaceous naphthoquinones in comparison with atovaquone targeting the selection of potential antimalarial candidates.

The natural naphthoquinones lapachol, α- and ß-lapachone are found in Bignoniaceous Brazilian plant species of the Tabebuia genus (synonym Handroanthus) and are recognized for diverse bioactivities, including as antimalarial. The aim of the present work was to perform in silico, in vitro and in vivo studies to evaluating the antimalarial potential of these three naphthoquinones in comparison with atovaquone, a synthetic antimalarial. The ADMET properties of these compounds were predicted in silico by the preADMET program. The in vitro toxicity assays were experimentally determined in immortalized and tumoral cells from different organs. In vivo acute oral toxicity was also evaluated for lapachol. Several favorable pharmacokinetics data were predicted although, as expected, high cytotoxicity was experimentally determined for ß-lapachone. Lapachol was not cytotoxic or showed low cytotoxicity to all of the cells assayed (HepG2, A549, Neuro 2A, LLC-PK1, MRC-5), it was nontoxic in the acute oral test and disclosed the best parasite selectivity index in the in vitro assays against chloroquine resistant Plasmodium falciparum W2 strain. On the other hand, α- and ß-lapachone were more potent than lapachol in the antiplasmodial assays but with low parasite selectivity due to their cytotoxicity. The diversity of data here reported disclosed lapachol as a promising candidate to antimalarial drug development.