Squaramides (SQA) were reported as potent antituberculosis drugs through inhibition of the mycobacterial enzyme adenosine triphosphate (ATP) synthase, which is critical for ATP synthesis. However, squaramide compounds showed high metabolic clearance (CL) despite their promising potency and selectivity. Herein, we describe lead optimization efforts to improve the potency and metabolic stability of previous lead 1f. Multiple squaramide analogues exhibited improved potency. The most potent analogue 20j expressed enhanced potency of 51 nM and moderate metabolic stability. SQA 6k displayed optimum balance of potency and metabolic stability both in vitro and in vivo. Notably, against bedaquiline-resistant Rv0678 mutants, a modest 2-fold increase in 6k minimum inhibitory concentration (MIC) was observed─reversed by complementation─versus a 16-fold shift in bedaquiline (BDQ) MIC. In a chronic tuberculosis (TB) mouse model, 6k coadministered with 1-aminobenzotriazole (ABT) exhibited bactericidal activity. These findings provide key strategies for improving potency and pharmacokinetic properties toward a tractable preclinical candidate.

Candida albicans, responsible for approximately 647,000 deaths annually, has been identified by the WHO as a priority pathogen for targeted antifungal drug development. To this end, we have screened microbial strains from the public outreach Swab and Send project for anti-Candida activity. This process identified Pseudomonas sp. SS1954.14 which displayed potent activity against Candida albicans, both on-agar and in a cell free supernatant assay. Whole-genome analysis identified this strain as Pseudomonas monsensis. It contains several biosynthetic gene clusters suggesting it can produce hydrogen cyanide, lokisin, pyoverdine, and colicin/carocin, which may contribute to the observed antifungal activity. However, an active compound was purified by preparative high-performance liquid chromatography and identified through high-resolution mass spectrometry as maculosin (cyclo-(L-Pro-L-Tyr)). This cyclic dipeptide has previously been found to possess antifungal activity, but the exact biosynthetic mechanism remains undetermined. Reporting this genome alongside the associated evidence of maculosin production represents a valuable resource for biosynthetic investigations.

IntroductionOnchocerciasis and lymphatic filariasis (LF) cause a significant global public health burden with more than 900 million individuals at risk and over 60 million people living with symptomatic manifestations caused by filarial diseases. Due to the importance of Wolbachia for the survival of adult filarial nematodes, anti-Wolbachia therapy has been validated as a safe macrofilaricidal treatment for LF and onchocerciasis. The A-WOL consortium was established with the goals of defining registered anti-Wolbachia antibiotics, as well as developing new drugs effective in a regimen of 7 days or less. We have previously shown autophagy has a core role in the regulation of Wolbachia populations across a diverse range of associations. In this study, we investigated the role of autophagy in the efficacy of anti-Wolbachia drugs.MethodsAutophagic flux was assessed in response to broad-spectrum anti-Wolbachia antibiotics (doxycycline, rifampicin, moxifloxacin, and sparfloxacin) and A-WOL candidate compounds (flubentylosin [TylAMac], AWZ1066S, and fusidic acid), compared with antibiotics with no activity against Wolbachia (levofloxacin, ciprofloxacin, amoxicillin, and streptomycin). Autophagy was quantified through LC3B-I to LC3B-II conversion and p62 degradation in insect cell lines (C6/36 and Sf9) and in Brugia malayi. The role of autophagy was evaluated using early- and late-stage inhibitors. The generation of ROS was measured to assess its contribution to autophagy activation. Finally, the viability of purified extracellular Wolbachia following drug exposure was determined using live/dead staining and reinfection assays.ResultsAll effective anti-Wolbachia compounds induced autophagic flux in insect cells and B. malayi, whereas ineffective antibiotics did not. Autophagy activation occurred in the absence of Wolbachia, was restricted to insect and nematode systems, and was not preceded by the generation of ROS. Only concentrations that induced autophagy resulted in effective Wolbachia depletion (of >90%), the empirical threshold of delivering the desired macrofilaricidal activity, and autophagy inhibition reduced the efficacy of the drugs. Exposure of purified extracellular Wolbachia to anti-Wolbachia drugs showed no impact on their viability, indicating that host processes are required for depletion.DiscussionThese results demonstrate the critical role of host autophagy in anti-wolbachia drug activity and a previously unrecognised host-directed mechanism in insect cells and B. malayi.

Objectives:Resistance to β-lactam antibiotics, including carbapenems, mediated by metallo-β-lactamases (MBLs), including the New Delhi metallo-β-lactamase (NDM) family, is increasing. No MBL inhibitors are currently approved for clinical use, with most reported MBL inhibitors acting as metal ion chelators at either the Zn(II) ion active site and/or in solution. The hexokinase inhibitor 3-bromopyruvate (3-BP) has been reported to inhibit NDM-1. This study investigates the ability of 3-BP to restore carbapenem activity against NDM-producing bacterial strains. Materials and methods:The ability of 3-BP to restore meropenem activity was assessed against carbapenem-resistant Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii strains, obtained from clinical and environmental isolates from Tanzania and Malawi. The mechanism of inhibition was investigated using mass spectrometry studies with both biologically relevant sulphur-containing compounds in addition to NDM-1 and NDM-5. Results and conclusions:3-BP selectively restored the antimicrobial activity of meropenem against resistant strains containing genes encoding NDM-1 or NDM-5 but did not restore the antimicrobial activity of amoxicillin against resistant strains containing genes encoding serine β-lactamases. Mass spectrometry studies with NDM-1 and NDM-5 support a mechanism involving covalent reaction of 3-BP with an active site cysteine residue. These results will promote work on the development of covalently reacting MBL inhibitors, a strategy that has been successful for inhibition of the nucleophilic serine β-lactamases.

Diazirines are versatile carbene precursors that are extensively used in biological target identification experiments. However, their photo-activation wavelength (ca. 365 nm) precludes their use in living organisms. Here we show that a reconceptualization of the diazirine hybridization state leads to conjugation of the diazirine motif to longer-wavelength chromophores. In a model diazirine–fluorene conjugate, we are able to achieve direct activation (and subsequent C–H insertion) with >450 nm light for the first time. Two-photon activation using red light is also achieved, suggesting the possibility to prepare new diazirine probes for conducting target identification experiments in deep tissue.

Aryl quinolone derivatives can target the cytochrome bc1 complex of Plasmodium falciparum, exhibiting excellent in vitro and in vivo antimalarial activity. However, their clinical development has been hindered due to their poor aqueous solubility profiles. In this study, a series of bioisosteres containing saturated heterocycles fused to a 4pyridone ring were designed to replace the inherently poorly soluble quinolone core in antimalarial quinolones with the aim to reduce it-it stacking interactions in the crystal packing solid state, and a synthetic route was developed to prepare these alternative core derivatives. One such novel derivate, F14, exhibited significant enhancements in both aqueous solubility (20 mu M) and lipophilicity (LogD 2.7), whilst retaining nanomolar antimalarial activity against the W2 strain of P. falciparum (IC50 = 235 nM). The pharmacokinetic studies reported, provide preliminary insights into the in vivo distribution and elimination of F14, while findings from single crystal X-ray diffraction experiment rationalized the enhanced solubility. Protein X-ray crystallography and in silico docking simulations provide insight into the potential mode of action within the cytochrome bc1 complex. These findings demonstrated the viability of this bioisostere replacement strategy and provided support for further exploration of in vivo efficacy in preclinical animal models and valuable insights for new drug design strategies in the fight against malaria.

Lymphatic filariasis and onchocerciasis are two major neglected tropical diseases that are responsible for causing severe disability in 50 million people worldwide, whilst veterinary filariasis (heartworm) is a potentially lethal parasitic infection of companion animals. There is an urgent need for safe, short-course curative (macrofilaricidal) drugs to eliminate these debilitating parasite infections. We investigated combination treatments of the novel anti-Wolbachia azaquinazoline small molecule, AWZ1066S, with benzimidazole drugs (albendazole or oxfendazole) in up to four different rodent filariasis infection models: Brugia malayi—CB.17 SCID mice, B. malayi—Mongolian gerbils, B. pahangi—Mongolian gerbils, and Litomosoides sigmodontis—Mongolian gerbils. Combination treatments synergised to elicit threshold (>90%) Wolbachia depletion from female worms in 5 days of treatment, using 2-fold lower dose-exposures of AWZ1066S than monotherapy. Short-course lowered dose AWZ1066S-albendazole combination treatments also delivered partial adulticidal activities and/or long-lasting inhibition of embryogenesis, resulting in complete transmission blockade in B. pahangi and L. sigmodontis gerbil models. We determined that short-course AWZ1066S-albendazole co-treatment significantly augmented the depletion of Wolbachia populations within both germline and hypodermal tissues of B. malayi female worms and in hypodermal tissues in male worms, indicating that anti-Wolbachia synergy is not limited to targeting female embryonic tissues. Our data provides pre-clinical proof-of-concept that sub-seven-day combinations of rapid-acting novel anti-Wolbachia agents with benzimidazole anthelmintics are a promising curative and transmission-blocking drug treatment strategy for filarial diseases of medical and veterinary importance.

AWZ1066S has been developed as a potential treatment for the neglected tropical diseases lymphatic filariasis and onchocerciasis. AWZ1066S targets the Wolbachia bacterial endosymbiont present in the causative nematode parasites. This phase 1, first-in-human study aimed to assess the safety and pharmacokinetics of AWZ1066S in healthy human participants. In a randomized double-blind, placebo-controlled, single ascending dose study, healthy adults received a single oral dose of AWZ1066S (or placebo) and were followed up for 10 days. The planned single doses of AWZ1066S ranged from 100 to 1600 mg, and each dose was administered to a cohort of 8 participants (6 AWZ1066S and 2 placebo). In total 30 people participated, 18 (60%) female, median age 30.0 years (minimum 20, maximum 61). The cohorts administered 100, 200, 300, and 400 mg of AWZ1066S progressed unremarkably. After single 700-mg doses all 4 participants developed symptoms of acute gastritis and transient increases in liver enzymes. The severity of these adverse events ranged from mild to severe, with 1 participant needing hospital admission. Pharmacokinetic analysis indicated that AWZ1066S is rapidly absorbed with predictable pharmacokinetics. In conclusion, safety concerns prevented this study from reaching the human exposures needed for AWZ1066S to be clinically effective against lymphatic filariasis and onchocerciasis.

The misuse and abuse of antibiotics have led to the increase of drug resistance and the emergence of multi-drug resistant bacteria. Therefore, it is an urgent need to develop novel antimicrobial agents to address this problem. Natural products (NPs) could provide an effective strategy for the discovery of drug due to their wide range of source and biological activities. Ursolic acid (UA) is a naturally occurring compound known for its wide range of biological properties. In this study, a series of UA derivatives were rationally designed and synthesized by incorporating antibacterial potential fragments of benzenesulfonamide and indole, with the aim of obtaining novel UA derivatives for the treatment of bacterial infections. Based on the preliminary screening, UA derivatives 27 (yield of 26 %), containing 4-chlorobenzenesulfonamide and 6-carboxyindole pharmacophores, as well as 34 (yield of 42 %), containing 4-carboxybenzenesulfonamide and unsubstituted indole pharmacophores, were identified as promising antibacterial agents against Staphylococcus aureus, especially for methicillin-resistant Staphylococcus aureus (MRSA), possessing MICs of 1 μM. Furthermore, both of them also displayed low hemolytic activity, non-resistance, and low-toxicity to mammalian cells. In addition, further mechanistic studies revealed that 27 and 34 were able to inhibit and eliminate MRSA biofilm formation, affecting the permeability of bacterial cell membrane, leading to increase intracellular reactive oxygen species (ROS) and ultimately inducing bacterial death. Notably, 27 and 34 also showed promising in vivo efficacy against MRSA in a mouse wound model. These results suggested that 27 and 34 should have promising applications against MRSA infection.

The emergence of Plasmodium parasite resistance to current front-line antimalarial treatments poses a serious threat to global malaria control and highlights the necessity for the development of therapeutics with novel targets and mechanisms of action. Plasmepsins IX and X (PMIX/PMX) have been recognised as highly promising targets in Plasmodium due to their contribution to parasite's pathogenicity. Recent research has demonstrated that dual PMIX/PMX inhibition results in the impairment of multiple parasite's life cycle stages, which is an important feature in drug resistance prevention. Herein we report novel hydroxyethylamine photoaffinity labelling (PAL) probes, designed for PMIX/PMX target engagement and proteomics experiments in Plasmodium parasites. The prepared probes have both a photoreactive group (diazirine or benzophenone) for covalent attachment to target proteins, and a terminal alkyne handle allowing their use in bioorthogonal ligation. One of the synthesised benzophenone probes was shown to be highly promising as demonstrated by its outstanding antimalarial potency (IC50 = 15 nM versus D10 P. falciparum) and its inhibitory effect against PfPMX in an enzymatic assay. Molecular docking and molecular dynamics studies show that the inclusion of the benzophenone and alkyne handle does not alter the binding mode compared to the parent compound. The photoaffinity probe can be used in future chemical proteomics studies to allow hydroxyethylamine drug scaffold target identification and validation in Plasmodium. We expect our findings to act as a tool for future investigations on PMIX/PMX inhibition in antimalarial drug discovery.

The rapid emergence of antibiotic resistance and the scarcity of novel antibacterial agents have necessitated an urgent pursuit for the discovery and development of novel antibacterial agents against multidrug-resistant bacteria. This study involved the design and synthesis of series of novel indole-benzosulfonamide oleanolic acid (OA) derivatives, in which the indole and benzosulfonamide pharmacophores were introduced into the OA skeleton semisynthetically. These target OA derivatives show antibacterial activity against Staphylococcus strains in vitro and in vivo. Among them, derivative c17 was the most promising antibacterial agent while compared with the positive control of norfloxacin, especially against methicillin-resistant Staphylococcus aureus (MRSA) in vitro. In addition, derivative c17 also showed remarkable efficacy against MRSA-infected murine skin model, leading to a significant reduction of bacterial counts during this in vivo study. Furthermore, some preliminary studies indicated that derivative c17 could effectively inhibit and eradicate the biofilm formation, disrupt the integrity of the bacterial cell membrane. Moreover, derivative c17 showed low hemolytic activity and low toxicity to mammalian cells of NIH 3T3 and HEK 293T. These aforementioned findings strongly support the potential of novel indole-benzosulfonamide OA derivatives as anti-MRSA agents.

Current treatments for diabetic wounds have some curative effect, but the process is complicated and lack user-friendly wound dressings. Nanozymes have gained significant attention for wound healing due to their striking merits. Herein, we have developed a novel sprayable tannin acid-cobalt coordination nanozyme (TACo) for diabetic wound healing. TACo nanozyme offers a convenient and efficient methods by spraying directly onto wounds surface, reducing infection risk by avoiding direct contact. Notably, its antioxidant properties contribute to scavenging the reactive oxygen species (ROS), alleviating oxidative stress and inflammation of wound microenvironment. Additionally, TACo nanozyme could promote cell survival and multiplication, which is crucial for the wound healing process. Importantly, TACo nanozyme facilitates angiogenesis by enhancing cell viability, migration, and tube formation. The unique coordination between metal and phenolic components confers pH-responsive cobalt ion and TA release properties, avoiding secondary damage during the wound cleaning. This unique composition seamlessly integrates photothermal antibacterial therapy, inflammatory microenvironment management, supporting for angiogenesis, and effective promotion of extracellular matrix production sequentially by harnessing the acidic pH environment of diabetic wounds. In conclusion, the development of a sprayable TACo nanozyme presents a promising therapeutic approach for the treatment of diabetic wounds, addressing the complexities of current treatments and providing a user-friendly application method.

Anti-Wolbachia therapy has been clinically proven to be a safe approach for the treatment of onchocerciasis and lymphatic filariasis. AWZ1066S, a first-in-class highly specific anti-Wolbachia drug candidate developed for a short-course treatment of human filariasis, has advanced into clinical development. An improved, cost-efficient, and scalable process for the manufacture of this clinical candidate is described. Presented herein is the process development work for the active pharmaceutical ingredient (API) and its two key starting materials [2-(trifluoromethyl)-3-pyridyl]methanamine and (S)-3-methylmorpholine, starting from 2,4-dichloropyrido[2,3-d]pyrimidine, which is capable of delivering high-purity (>99%) API consistently. The optimized production route was used in the manufacture of the clinical candidate at the kilogram scale to support the ongoing clinical development.

Introduction: Fusidic acid (FA) has been widely applied in the clinical prevention and treatment of bacterial infections. Nonetheless, its clinical application has been limited due to its narrow antimicrobial spectrum and some side effects.Purpose: Therefore, it is necessary to explore the structure–activity relationships of FA derivatives as antibacterial agents to develop novel ones possessing a broad antimicrobial spectrum.Methods and result: First, a pharmacophore model was established on the nineteen FA derivatives with remarkable antibacterial activities reported in previous studies. The common structural characteristics of the pharmacophore emerging from the FA derivatives were determined as those of six hydrophobic centers, two atom centers of the hydrogen bond acceptor, and a negative electron center around the C-21 field. Then, seven FA derivatives have been designed according to the reported structure–activity relationships and the pharmacophore characteristics. The designed FA derivatives were mapped on the pharmacophore model, and the Qfit values of all FA derivatives were over 50 and FA-8 possessed the highest value of 82.66. The molecular docking studies of the partial target compounds were conducted with the elongation factor G (EF-G) of S. aureus. Furthermore, the designed FA derivatives have been prepared and their antibacterial activities were evaluated by the inhibition zone test and the minimum inhibitory concentration (MIC) test. The derivative FA-7 with a chlorine group as the substituent group at C-25 of FA displayed the best antibacterial property with an MIC of 3.125 µM. Subsequently, 3D-QSAR was carried on all the derivatives by using the CoMSIA mode of SYBYL-X 2.0.Conclusion: Hence, a computer-aided drug design model was developed for FA, which can be further used to optimize FA derivatives as highly potent antibacterial agents.

Two series of novel sophoridine derivatives were designed, synthesized, and evaluated for their anti-mosquito activity. SOP-2g, SOP-2q, and SOP-2r exhibited potential larvicidal activity against Aedes albopictus larva with LC50 values of 330.98, 430.53, and 411.09 ppm, respectively. Analysis of structure-activity relationships indicated that the oxime ester group was beneficial for improving the larvicidal biological activity, whereas the long-chain aliphatic group and fused-ring group were introduced. Furthermore, the larvicidal mechanism was also investigated based on the inhibition assay of acetylcholinesterase (AChE) and the morphological observation of dead larva treated with derivatives. Results indicated that the AChE inhibitory activity of the preferred three derivatives were 63.16%, 46.67%, and 35.11%, respectively, at 250 ppm concentration. Additionally, morphological evidence demonstrated that SOP-2q and SOP-2r induced changes in the larva's intestinal cavity, caudal gill, and tail, thereby displaying larvicidal action against Ae. albopictus together with AChE inhibition. Therefore, this study implied that sophoridine and its novel derivatives could be used to control the population of mosquito larva, which may also be effective alkaloids to reduce the mosquito population density.

ABSTRACT Use of experimental cats and dogs in veterinary heartworm preclinical drug research is increasing. As a potential alternative primary in vivo heartworm preventative drug screen, we assessed lymphopenic mice with ablation of the interleukin-2/7 common gamma chain (γc) as susceptible to the larval development phase of D. immitis . Non-obese diabetic (NOD) Severe Combined ImmunoDeficient (SCID)γc -/- (NSG / NXG) mice consistently yielded viable D. immitis larvae at 2-4 weeks post-infection across multiple experiments, different batches of infectious larvae inoculates, different isolates of D. immitis and at independent laboratories. Mice did not display any overt clinical signs associated with infection up to 4 weeks. Developing larvae were found in subcutaneous and muscle fascia tissues, the natural site of this stage of heartworm in dogs. Larvae retrieved from NSG / NXG mice were mid-L4 stage of development. Compared with 14-day in vitro propagated larvae, in vivo derived L4 were significantly larger and contained expanded Wolbachia endobacteria titres, determined by QPCR and Fluorescent in situ Hybridisation (FISH). We established an ex vivo 6-day L4 paralytic screening system against nematodicidal agents (moxidectin, levamisole) which highlighted discrepancies in relative drug sensitivities in comparison with in vitro reared L4 D. immitis. We demonstrated effective depletion of Wolbachia by 70-90% in D. immitis L4 following 2-7 day oral in vivo exposures of NSG / NXG infected mice with doxycycline or the rapid-acting investigational anti- Wolbachia drug, AWZ1066S. We validated the NSG / NXG mouse model as a filaricide drug screen by in vivo treatments with single injections of moxidectin, which mediated 60-88% reduction in L4 larvae at 14-28 days. Future adoption of the mouse model as a first-line efficacy screen will benefit end-user laboratories conducting research and development of novel heartworm preventatives via increased access, rapid turnaround and reduced costs whilst simultaneously decreasing need for experimental cat or dog use.

Mycobacterium tuberculosis cytochrome bd quinol oxidase (cyt bd), the alternative terminal oxidase of the respiratory chain, has been identified as playing a key role during chronic infection and presents a putative target for the development of novel antitubercular agents. Here, we report confirmation of successful heterologous expression of M. tuberculosis cytochrome bd. The heterologous M. tuberculosis cytochrome bd expression system was used to identify a chemical series of inhibitors based on the 2-aryl-quinolone pharmacophore. Cytochrome bd inhibitors displayed modest efficacy in M. tuberculosis growth suppression assays together with a bacteriostatic phenotype in time-kill curve assays. Significantly, however, inhibitor combinations containing our front-runner cyt bd inhibitor CK-2-63 with either cyt bcc-aa3 inhibitors (e.g., Q203) and/or adenosine triphosphate (ATP) synthase inhibitors (e.g., bedaquiline) displayed enhanced efficacy with respect to the reduction of mycobacterium oxygen consumption, growth suppression, and in vitro sterilization kinetics. In vivo combinations of Q203 and CK-2-63 resulted in a modest lowering of lung burden compared to treatment with Q203 alone. The reduced efficacy in the in vivo experiments compared to in vitro experiments was shown to be a result of high plasma protein binding and a low unbound drug exposure at the target site. While further development is required to improve the tractability of cyt bd inhibitors for clinical evaluation, these data support the approach of using small-molecule inhibitors to target multiple components of the branched respiratory chain of M. tuberculosis as a combination strategy to improve therapeutic and pharmacokinetic/pharmacodynamic (PK/PD) indices related to efficacy.

IntroductionDirofilariasis, including heartworm disease, is a major emergent veterinary parasitic infection and a human zoonosis. Currently, experimental infections of cats and dogs are used in veterinary heartworm preclinical drug research.MethodsAs a refined alternative in vivo heartworm preventative drug screen, we assessed lymphopenic mouse strains with ablation of the interleukin-2/7 common gamma chain (γc) as susceptible to the larval development phase of Dirofilaria immitis.ResultsNon-obese diabetic (NOD) severe combined immunodeficiency (SCID)γc−/− (NSG and NXG) and recombination-activating gene (RAG)2−/−γc−/− mouse strains yielded viable D. immitis larvae at 2–4 weeks post-infection, including the use of different batches of D. immitis infectious larvae, different D. immitis isolates, and at different laboratories. Mice did not display any clinical signs associated with infection for up to 4 weeks. Developing larvae were found in subcutaneous and muscle fascia tissues, which is the natural site of this stage of heartworm in dogs. Compared with in vitro-propagated larvae at day 14, in vivo-derived larvae had completed the L4 molt, were significantly larger, and contained expanded Wolbachia endobacteria titres. We established an ex vivo L4 paralytic screening system whereby assays with moxidectin or levamisole highlighted discrepancies in relative drug sensitivities in comparison with in vitro-reared L4 D. immitis. We demonstrated effective depletion of Wolbachia by 70%−90% in D. immitis L4 following 2- to 7-day oral in vivo exposures of NSG- or NXG-infected mice with doxycycline or the rapid-acting investigational drug, AWZ1066S. We validated NSG and NXG D. immitis mouse models as a filaricide screen by in vivo treatments with single injections of moxidectin, which mediated a 60%−88% reduction in L4 larvae at 14–28 days.DiscussionFuture adoption of these mouse models will benefit end-user laboratories conducting research and development of novel heartworm preventatives via increased access, rapid turnaround, and reduced costs and may simultaneously decrease the need for experimental cat or dog use.

The potential for essential oils (EOs) to act as a natural alternative for controlling mosquito populations has aroused extensive interest in recent years. Four EOs (Thymus vulgaris (thyme), Foeniculum vulgare (fennel), Citrus medica (lime), and Camellia sinensis (green tea)) were selected in our previous larvicidal screening assays against Aedes albopictus. In this study, the active constituents of each EO were identified. Next, their acetylcholinesterase (AChE) inhibitory activity was evaluated, and the binding mode was studied by molecular docking. To improve EO stability and duration of activity, nanoemulsions were prepared and characterized. Thirteen constituents of the four EOs at the 100 ppm level led to 100% mortality of the larvae, the four most active constituents being α-hexyl cinnamaldehyde (LC50 = 24.0 ± 0.3 ppm), limonene (LC50 = 34.1 ± 0.7 ppm), α-terpinene (LC50 = 50.0 ± 0.5 ppm), and carvacrol (LC50 = 54.7 ± 0.6 ppm). These compounds, with the exception of α-hexyl cinnamaldehyde, also showed potent AChE inhibitory activity. Nanoemulsions with droplet size of 56.8–224.9 nm were prepared by emulsification at a 1:1 (v/v) ratio of EO and Tween 80. These EO nanoemulsions showed equally potent larvicidal activity as EOs themselves; moreover, they showed better stability and were active for longer time. These results suggest that natural EOs and their constituents have potential as larvicides for mosquito vector control and that nanoemulsification is an effective method for improving their physicochemical properties and efficacy.

18 beta-glycyrrhetinic acid (GA) is a well-known natural compound of oleanane-type triterpene and is found possessing antimicrobial and anti-inflammatory properties. Nonetheless, its relatively low bioactivity restricts its potential in pharmaceutical applications. To maximize the potential use of this natural herbal compound as antimicrobial and anti-inflammatory agents, the rational modification of GA to enhance its pharmacological activity with low toxicity and to understand the mechanism of action is critically essential. We reported herein the design and synthesis of a series of new GA derivatives. The antimicrobial activities of these new compounds were evaluated by inhibition zone test and minimum inhibitory concentration (MIC) assay. In addition, the anti-inflammatory activity was evaluated by LPS induced BV2 cells inflammation model and 12-O-tetradecanoyl phorbol-13-acetate (TPA) induced ear inflammation mice model. It was found that the derivatives functionalized with a di-substituted phenyl group at the 2-position of GA generally displayed high antimicrobial activity against Gram-positive bacteria (MIC down to 2.5 mu M) and potent anti-inflammatory effects (inhibition of NO production up to 55%, comparable to dexamethasone). The in vitro and in vivo results also showed that GA-O-02 and GA-O-06 exert their anti-inflammatory activities through downregulation of NO, pro-inflammatory cytokines and chemokines (IL-1 beta, IL-6, IL-12, TNF-alpha, MCP-1 and MIP-1 alpha) and upregulation of anti-inflammatory cytokines (IL 10). The anti-inflammatory mechanism may involve the inhibition of NF-kappa B, MAPKs and PI3K/Akt related inflammatory signaling pathways and activation of Nrf2/HO-1 signaling pathway. The results demonstrated that GA-O-02 and GA-O-06 possess great application potential as potent antimicrobial and anti-inflammatory agents.