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The development of parasite resistance to both artemisinin derivatives and their partner drugs jeopardizes the effectiveness of the artemisinin combination therapy. Thus, the discovery of new antimalarial drugs, with new mechanisms of action, is urgently needed. We recently disclosed that β-carboline 1a was orally efficacious in Plasmodium berghei -infected mice and that it showed low cross-resistance between susceptible Plasmodium falciparum and four different drug-resistant strains. In this report, we describe the synthesis and in vitro antimalarial evaluation of 91 new derivatives of 1a . The asexual blood stage growth inhibition data show a clear preference for a 3,4-dihalogenated, 3,5-dihalogenated, 3,4,5-trichloro-, or 4-trifluoromethyphenyl ring at the C1-position. The most potent compound, 3,4,5-trichlorophenyl-substituted 42a , is twice as potent as 1a . Six potent analogues were assessed for their drug-like properties, and four of these were subjected to in vitro barcoded cross-resistance profiling. Compounds 1a , 1m , 42a , and 42m showed no cross-resistance to 32 resistance mutations on the Dd2 genetic background and 10 resistance mutations on the 3D7 genetic background. These data suggest that compounds in this scaffold possess a novel mechanism of antimalarial action.

Drug-resistant Neisseria gonorrhoeae represents a major threat to public health; without new effective antibiotics, untreatable gonococcal infections loom as a real possibility. In a previous drug-repurposing study, we reported that salicylic acid had good potency against azithromycin-resistant N. gonorrhoeae. We now report that the anti-gonococcal activity in this scaffold is easily lost by inopportune substitution, but that select substituted naphthyl analogs (3b, 3o and 3p) have superior activity to salicylic acid itself. Furthermore, these compounds retained potency against multiple ceftriaxone- and azithromycin-resistant strains, exhibited rapid bactericidal activity against N. gonorrhoeae, and showed high tolerability to mammalian cells (CC 50  > 128 µg/mL). Promisingly, these compounds also show very weak growth inhibition of commensal vaginal bacteria., (© 2023. Springer Nature Limited.)

With the pressing antibiotic resistance pandemic, antivirulence has been increasingly explored as an alternative strategy against bacterial infections. The bacterial type IV pilus (T4P) is a well-documented virulence factor and an attractive target for small molecules for antivirulence purposes. The PilB ATPase is essential for T4P biogenesis because it catalyzes the assembly of monomeric pilins into the polymeric pilus filament. Here, we describe the identification of two PilB inhibitors by a high-throughput screen (HTS) in vitro and their validation as effective inhibitors of T4P assembly in vivo . We used Chloracidobacterium thermophilum PilB as a model enzyme to optimize an ATPase assay for the HTS. From a library of 2,320 compounds, benserazide and levodopa, two approved drugs for Parkinson's disease, were identified and confirmed biochemically to be PilB inhibitors. We demonstrate that both compounds inhibited the T4P-dependent motility of the bacteria Myxoccocus xanthus and Acinetobacter nosocomialis. Additionally, benserazide and levodopa were shown to inhibit A. nosocomialis biofilm formation, a T4P-dependent process. Using M. xanthus as a model, we showed that both compounds inhibited T4P assembly in a dose-dependent manner. These results suggest that these two compounds are effective against the PilB protein in vivo. The potency of benserazide and levodopa as PilB inhibitors both in vitro and in vivo demonstrate potentials of the HTS and its two hits here for the development of anti-T4P chemotherapeutics. IMPORTANCE Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens. Here, we report the development and implementation of a method to identify anti-T4P chemicals from compound libraries by high-throughput screen. This led to the identification and validation of two T4P inhibitors both in the test tubes and in bacteria. The discovery and validation pipeline reported here as well as the confirmation of two anti-T4P inhibitors provide new venues and leads for the development of chemotherapeutics against antibiotic-resistant infections.

The tetrahydro-β-carboline scaffold has proven fertile ground for the discovery of antimalarial agents (e.g., MMV008138 ( 1 ) and cipargamin ( 2 )). Similarity searching of a publicly disclosed collection of antimalarial hits for molecules resembling 1 drew our attention to N2-acyl tetrahydro-β-carboline GNF-Pf-5009 ((±)- 3b ). Compound purchase, "analog by catalog", and independent synthesis of hits indicated the benzofuran-2-yl amide portion was required for in vitro efficacy against P. falciparum . Preparation of pure enantiomers demonstrated the pharmacological superiority of ( R )- 3b . Synthesis and evaluation of D- and F-ring substitution variants and benzofuran isosteres indicated a clear structure-activity relationship. Ultimately ( R )- 3b was tested in Plasmodium berghei -infected mice; unfavorable physicochemical properties may be responsible for the lack of oral efficacy., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

Virtual ligand screening of a publicly available database of antimalarial hits using a pharmacophore derived from antimalarial MMV008138 identified TCMDC-140230, a tetrahydro-β-carboline amide, as worthy of exploration. All four stereoisomers of this structure were synthesized, but none potently inhibited growth of the malaria parasite Plasmodium falciparum . Interestingly, 7e , a minor byproduct of these syntheses, proved to be potent in vitro against P. falciparum and was orally efficacious (40 mg/kg) in an in vivo mouse model of malaria., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

Background: Varroa destructor is among the greatest threats to honey bee health worldwide. Acaricides used to control Varroa are becoming increasingly ineffective due to resistance issues, prompting the need for new compounds that can be used for control purposes. Ideally, such compounds would exhibit high toxicity to Varroa while maintaining relatively low toxicity to bees and beekeepers. We characterized the lethal concentrations (LC 50 ) of amitraz, matrine, FlyNap®, the experimental carbamates 2-((2-ethylbutyl)thio)phenyl methylcarbamate (1) and 2-(2-ethylbutoxy)phenyl methylcarbamate (2), and dimethoate (positive control) for Varroa using a glass vial assay. The test compounds also were applied to honey bees using an acute contact toxicity assay to determine the adult bee LD 50 for each compound., Results: Amitraz was the most toxic compound to Varroa, but carbamate 2 was nearly as active (within 2-fold) and the most selective due to its lower bee toxicity, demonstrating its promise as a Varroa control. While carbamate 1 was less toxic to honey bees than was amitraz, it was also 4.7-fold less toxic to the mites. Both matrine and FlyNap® were relatively ineffective at killing Varroa and were moderately toxic to honey bees., Conclusion: Additional testing is required to determine if carbamate 2 can be used as an effective Varroa control. As new chemical treatments are identified, it will be necessary to determine how they can be utilized best alongside other control techniques as part of an integrated pest management program. © 2021 Society of Chemical Industry., (© 2021 Society of Chemical Industry.)

Neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease, are devastating diseases in the elderly world, which are closely associated with progressive neuronal loss induced by a variety of genetic and/or environmental factors. Unfortunately, currently available treatments for neurodegenerative disorders can only relieve the symptoms but not modify the pathological processes. Over the past decades, our group by collaborating with Profs. Yuan-Ping Pang and Paul R. Carlier has developed three series of homo/hetero dimeric acetylcholinesterase inhibitors derived from tacrine and/or huperzine A. The representative dimers bis(3)-Cognitin (B3C), bis(12)-hupyridone, and tacrine(10)-hupyridone might possess disease-modifying effects through the modulation of N-methyl-d-aspartic acid receptors, the activation of myocyte enhancer factor 2D gene transcription, and the promotion of neurotrophic factor secretion. In this review, we summarize that the representative dimers, such as B3C, provide neuroprotection against a variety of neurotoxins via multiple targets, including the inhibitions of N-methyl-d-aspartic acid receptor with pathological-activated potential, neuronal nitric oxide synthase, and β-amyloid cascades synergistically. More importantly, B3C might offer disease-modifying potentials by activating myocyte enhancer factor 2D transcription, inducing neuritogenesis, and promoting the expressions of neurotrophic factors in vitro and in vivo. Taken together, the novel dimers might offer synergistic disease-modifying effects, proving that dimerization might serve as one of the strategies to develop new generation of therapeutics for neurodegenerative disorders., (© 2021 International Society for Neurochemistry.)

Alzheimer's disease (AD) is a neurodegenerative disorder with multiple pathological features. Therefore, a multitarget-directed ligands (MTDLs) strategy has been developed to treat AD. We have previously designed and synthesized dimeric tacrine(10)-hupyridone (A10E), a novel tacrine derivative with acetylcholinesterase (AChE) inhibition and brain-derived neurotrophic factor (BDNF) activation activity, by linking tacrine and a fragment of huperzine A. However, it was largely unknown whether A10E could act on other AD targets and produce cognitive-enhancing ability in AD animal models. In this study, A10E could prevent cognitive impairments in APP/PS1 transgenic mice and β-amyloid (Aβ) oligomers-treated mice, with higher potency than tacrine and huperzine A. Moreover, A10E could effectively inhibit Aβ production and deposition, alleviate neuroinflammation, enhance BDNF expression, and elevate cholinergic neurotransmission in vivo . At nanomolar concentrations, A10E could inhibit Aβ oligomers-induced neurotoxicity via the activation of tyrosine kinase receptor B (TrkB)/Akt pathway in SH-SY5Y cells. Furthermore, Aβ oligomerization and fibrillization could be directly disrupted by A10E. Importantly, A10E at high concentrations did not produce obvious hepatotoxicity. Our results indicated that A10E could produce anti-AD neuroprotective effects via the inhibition of Aβ aggregation, the activation of the BDNF/TrkB pathway, the alleviation of neuroinflammation, and the decrease of AChE activity. As MTDLs could produce additional benefits, such as overcoming the deficits of drug combination and enhancing the compliance of AD patients, our results also suggested that A10E might be developed as a promising MTDL lead for the treatment of AD.

The antimalarial candidate MMV008138 (1a) is of particular interest because its target enzyme (IspD) is absent in human. To achieve higher potency, and to probe for steric demand, a series of analogs of 1a were prepared that featured methyl-substitution of the B- and C-rings, as well as ring-chain transformations. X-ray crystallography, NMR spectroscopy and calculation were used to study the effects of these modifications on the conformation of the C-ring and orientation of the D-ring. Unfortunately, all the B- and C-ring analogs explored lost in vitro antimalarial activity. The possible role of steric effects and conformational changes on target engagement are discussed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Antimalarial candidates possessing novel mechanisms of action are needed to control drug resistant Plasmodium falciparum. We were drawn to Malaria Box compound 1 (MMV665831) by virtue of its excellent in vitro potency, and twelve analogs were prepared to probe its structure-activity relationship. Modulation of the diethyl amino group was fruitful, producing compound 25, which was twice as potent as 1 against cultured parasites. Efforts were made to modify the phenolic Mannich base functionality of 1, to prevent formation of a reactive quinone methide. Homologated analog 28 had reduced potency relative to 1, but still inhibited growth with EC 50  ≤ 200 nM. Thus, the antimalarial activity of 1 does not derive from quinone methide formation. Chemical stability studies on dimethyl analog 2 showed remarkable hydrolytic stability of both the phenolic Mannich base and ethyl ester moieties, and 1 was evaluated for in vivo efficacy in P. berghei-infected mice (40 mg/kg, oral). Unfortunately, no reduction in parasitemia was seen relative to control. These results are discussed in the context of measured plasma and hepatocyte stabilities, with reference to structurally-related, orally-efficacious antimalarials., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Nucleophilic ring opening of cyclohexene oxides is known to proceed preferentially through the trans-diaxial pathway (the Fürst-Plattner rule). This preference, however, is not absolute, and can be affected by substituents on the cyclohexene oxide ring, as illustrated by LiAlH4 ring-opening of the cis- and trans-isomers of 4-t-butyl- and 3-methylcyclohexene oxide (cis- and trans-1, cis- and trans-2). We performed B3LYP/6-31+G*(PCM) geometry optimizations to locate the chair-like and twist-boat-like transition structures for the hydride attacks on the pseudoaxial and pseudoequatorial conformers of these epoxides. Our calculations are consistent with the experimental observation of effective Fürst-Plattner control of AlH4--opening of cis-1, trans-1, and cis-2, but low selectivity in ring-opening of trans-2. Our data at B3LYP/6-31+G*(PCM) suggests this reduction in selectivity is due to a diminished pseudoequatorial preference of the 3-methyl group in trans-2 relative to that in cis-2. The two calculated chair-like transition structures for hydride opening of trans-2 differ in activation energy free energy (ΔΔG‡) by only 0.4 kcal mol-1. Thus, these calculations account for the reduced regioselectivity of ring opening seen for trans-2 by AlH4- and other nucleophiles.

The Pictet-Spengler reaction of tryptophan esters and aldehydes has been widely applied in natural product synthesis and medicinal chemistry. To date, the trans- or cis-configuration of 1,3-disubstituted tetrahydro-β-carbolines (THβCs) formed in this reaction has most often been assigned based on the relative 13 C chemical shifts of C1 and C3 in the diastereomers. Although the upfield shifts of C1 and C3 in trans-THβCs relative to cis-THβCs has been attributed to steric compression associated with the "γ-gauche" effect, we show that this effect is not borne out experimentally for other carbons that should suffer this same compression. Thus we developed a robust alternative method for stereochemical assignment based on 1 H NMR coupling constants (31 examples) and supported by extensive DFT-based conformational analysis and calculation of 1 H- 1 H coupling constants. DFT calculations of 13 C NMR chemical shifts also cast doubt upon the role of the "γ-gauche" effect on C1 and C3 chemical shifts in trans-THβCs.

New insecticides are needed for control of disease-vectoring mosquitoes and this research evaluates the activity of new carbamate acetylcholinesterase (AChE) inhibitors. Biochemical and toxicological characterization of carbamates based on the parent structure of terbam, 3- tert -butylphenyl methylcarbamate, was performed. In vitro enzyme inhibition selectivity ( Anopheles gambiae versus human) was assessed by the Ellman assay, as well as the lethality to whole insects by the World Health Organization (WHO) paper contact assay. Bromination at the phenyl C6 position increased inhibitory potency to both AChEs, whereas a 6-iodo substituent led to loss of potency, and both halogenations caused a significant reduction of mosquitocidal activity. Similarly, installation of a hexyl substituent at C6 drastically reduced inhibition of Ag AChE, but showed a smaller reduction in the inhibition of hAChE. A series of 4-carboxamido analogs of the parent compound gave reduced activity against Ag AChE and generally showed more activity against hAChE than Ag AChE. Replacement of the 3- t -buyl group with CF 3 resulted in poor anticholinesterase activity, but this compound did have measurable mosquitocidal activity. A series of methyl- and fluoro- analogs of 3-trialkylsilyl compounds were also synthesized, but unfortunately resulted in disappointing activity. Finally, a series of sulfenylated proinsecticides showed poor paper contact toxicity, but one of them had topical activity against adult female Anopheles gambiae . Overall, the analogs prepared here contributed to a better understanding of carbamate structure-activity relationships (SAR), but no new significant leads were generated.

Post-operative cognitive dysfunction (POCD) could cause short-term or long-term cognitive disruption lasting weeks or months after anesthesia and surgery in elderly. However, no effective treatment of POCD is currently available. Previous studies indicated that the enhancement of brain-derived neurotrophic factor (BDNF) expression, and the elevation the cholinergic system, might be effective to prevent POCD. In this study, we have discovered that tacrine(10)-hupyridone (A10E), a novel acetylcholinesterase (AChE) inhibitor derived from tacrine and huperzine A, could prevent surgery-induced short-term and long-term impairments of recognition and spatial cognition, as evidenced by the novel object recognition test and Morris water maze (MWM) tests, in aged mice. Moreover, A10E significantly increased the expression of BDNF and activated the downstream Akt and extracellular regulated kinase (ERK) signaling in the surgery-treated mice. Furthermore, A10E substantially enhanced choline acetyltransferase (ChAT)-positive area and decreased AChE activity, in the hippocampus regions of surgery-treated mice, indicating that A10E could prevent surgery-induced dysfunction of cholinergic system, possibly via increasing the synthesis of acetylcholine and the inhibition of AChE. In conclusion, our results suggested that A10E might prevent POCD via the activation of BDNF pathway and the inhibition of AChE, concurrently, in aged mice. These findings also provided a support that A10E might be developed as a potential drug lead for POCD.

The present study focused on the toxicity of the aphid anti-feedant flonicamid and its main metabolite, 4-trifluoromethylnicotinamide (TFNA-AM) to Aedes aegypti and Anopheles gambiae mosquitoes. The compounds were toxic to both species via topical application, resulting in un-coordinated locomotion and leg splaying, with a favorable An. gambiae LD 50 value of 35 ng/mg for TFNA-AM, but no significant lethality to Ae. aegypti at 10 μg/female. There was mild cross resistance in the Akron-kdr (Akdr) strain of An. gambiae. Both compounds were non-toxic to intact larvae (LC 50  > 300 ppm); however, headless Ae. aegypti larvae displayed spastic paralysis, with PC 50 values of 2-4 ppm, indicating that the cuticle is a significant barrier to penetration. TFNA-AM showed low mammalian toxicity, with an LD 50 of >2000 mg/kg in mice. Electrophysiological experiments showed larval Aedes muscle depolarization and Kv2 channel blocking activity that required near mM concentrations, suggesting that this potassium channel is not the main target for flonicamid nor its metabolite. However, TFNA-AM was a potent blocker of evoked body wall sensory discharge in dipteran larvae, suggesting that some component of the chordotonal organ system may be involved in its toxicity. Finally, flonicamid and TFNA-AM showed about 2-fold synergism of permethrin toxicity against An. gambiae adult females whose mechanism should become more clear once the mode of action of these compounds is better defined., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The widespread emergence of pyrethroid-resistant Anopheles gambiae has intensified the need to find new contact mosquitocides for indoor residual spraying and insecticide treated nets. With the goal of developing new species-selective and resistance-breaking acetylcholinesterase (AChE)-inhibiting mosquitocides, in this report we revisit the effects of carbamate substitution on aryl carbamates, and variation of the 1-alkyl group on pyrazol-4-yl methylcarbamates. Compared to aryl methylcarbamates, aryl dimethylcarbamates were found to have lower selectivity for An. gambiae AChE ( Ag AChE) over human AChE (hAChE), but improved tarsal contact toxicity to G3 strain An. gambiae . Molecular modeling studies suggest the lower species-selectivity of the aryl dimethylcarbamates can be attributed to a less flexible acyl pocket in Ag AChE relative to hAChE. The improved tarsal contact toxicity of the aryl dimethylcarbamates relative to the corresponding methylcarbamates is attributed to a range of complementary phenomena. With respect to the pyrazol-4-yl methylcarbamates, the previously observed low An. gambiae -selectivity of compounds bearing α-branched 1-alkyl groups was improved by employing β- and γ-branched 1-alkyl groups. Compounds 22a (cyclopentylmethyl), 21a (cyclobutylmethyl), and 26a (3-methylbutyl) offer 250-fold, 120-fold, and 96-fold selectivity, respectively, for inhibition of Ag AChE vs. hAChE. Molecular modeling studies suggests the high species-selectivity of these compounds can be attributed to the greater mobility of the W84 side chain in the choline-binding site of Ag AChE, compared to that of W86 in hAChE. Compound 26a has reasonable contact toxicity to G3 strain An. gambiae (LC 50 = 269 μg/mL) and low cross-resistance to Akron strain (LC 50 = 948 μg/mL), which bears the G119S resistance mutation., Competing Interests: Conflict of Interest: None of the authors have any conflict to report.

Malaria continues to be one of the deadliest diseases worldwide, and the emergence of drug resistance parasites is a constant threat. Plasmodium parasites utilize the methylerythritol phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are essential for parasite growth. Previously, we and others identified that the Malaria Box compound MMV008138 targets the apicoplast and that parasite growth inhibition by this compound can be reversed by supplementation of IPP. Further work has revealed that MMV008138 targets the enzyme 2- C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD) in the MEP pathway, which converts MEP and cytidine triphosphate (CTP) to cytidinediphosphate methylerythritol (CDP-ME) and pyrophosphate. In this work, we sought to gain insight into the structure-activity relationships by probing the ability of MMV008138 analogs to inhibit PfIspD recombinant enzyme. Here, we report PfIspD inhibition data for fosmidomycin (FOS) and 19 previously disclosed analogs and report parasite growth and PfIspD inhibition data for 27 new analogs of MMV008138. In addition, we show that MMV008138 does not target the recently characterized human IspD, reinforcing MMV008138 as a prototype of a new class of species-selective IspD-targeting antimalarial agents.

Malaria is a devastating disease in sub-Saharan Africa and is transmitted by the mosquito Anopheles gambiae. While indoor residual spraying of anticholinesterase insecticides has been useful in controlling the spread of malaria, widespread application of these compounds has led to the rise of an insecticide-resistant mosquito strain that harbors a G119S mutation in the nervous system target enzyme acetylcholinesterase. We demonstrate the atomic basis of insecticide resistance through structure determination of the G119S mutant acetylcholinesterase of An. gambiae in the ligand-free state and bound to a potent difluoromethyl ketone inhibitor. These structures reveal specific features within the active-site gorge distinct from human acetylcholinesterase, including an open channel at the base of the gorge, and provide a means for improving species selectivity in the rational design of improved insecticides for malaria vector control., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

New public health insecticides are urgently required to prevent the spread of vector-borne disease. With the goal of identifying new K + -channel-directed mosquitocides, analogs of the RH-5849 family of diacyl t-butylhydrazines were synthesized and tested for topical toxicity against adult Anopheles gambiae, the African vector of malaria. In total, 80N'-monoacyl and N, N'-diacyl derivatives of benzyl- and arylhydrazines were prepared. Three compounds (2bo, 2kb, 3ab) were identified that were more toxic than RH-5849 and RH-1266. The potencies of these compounds to block K + currents in An. gambiae and human Kv2.1 channels were assessed to address their possible mechanism of mosquitocidal action. Selectivity for inhibition of An. gambiae Kv2.1 vs human Kv2.1 did not exceed 3-fold. Furthermore, no correlation was seen between the potency of insecticidal action and K + channel blocking potency. These observations, combined with the minimal knockdown seen with 2bo near its LD 50 value, suggests a mode of action outside of the nervous system., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Potential targets for new vector control insecticides are nerve and muscle potassium channels. In this study, the activities of known potassium channel blockers (4-aminopyridine, quinidine, and tetraethylammonium) and the insecticide propoxur were compared to three experimental catechols and several other compounds against Anopheles gambiae and Aedes aegypti mosquitoes. Experimental catechol 1 was the most toxic experimental compound in all of the mortality assays conducted, but was at least 100-fold and 39-fold less toxic than propoxur against Ae. aegypti and An. gambiae, respectively. Injection treatment and synergist (piperonyl butoxide) bioassays found that catechol toxicity was not unduly impacted by cuticular transport or oxidative metabolism. Electrophysiological studies showed a decrease in amplitude of evoked muscle contractions, along with an increase in twitch duration at concentrations that increased basal muscle tension (mM). High concentration effects on basal muscle tension were matched by complete depolarization of the muscle membrane potential. Effects on muscle physiology and blockage of Kv2.1 potassium channels in patch clamp experiments were generally consistent with in vivo toxicity, except for 4-aminopyridine, which suggest the involvement of other potassium channel subtypes. Extensive melanization of Anopheles larvae, but not Aedes larvae, occurred from exposure to catechol compounds. Interaction with the phenol oxidase system within insects may be the cause of this melanization, but any contribution to toxicity requires further investigation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Great reductions in malaria mortality have been accomplished in the last 15 years, in part due to the widespread roll-out of insecticide-treated bednets across sub-Saharan Africa. To date, these nets only employ pyrethroids, insecticides that target the voltage-gated sodium ion channel of the malaria vector, Anopheles gambiae. Due to the growing emergence of An. gambiae strains that are resistant to pyrethroids, there is an urgent need to develop new public health insecticides that engage a different target and possess low mammalian toxicity. In this review, we will describe efforts to develop highly species-specific and resistance-breaking inhibitors of An. gambiae acetylcholinesterase (AgAChE). These efforts have been greatly aided by advances in knowledge of the structure of the enzyme, and two major inhibitor design strategies have been explored. Since AgAChE possesses an unpaired Cys residue not present in mammalian AChE, a logical strategy to achieve selective inhibition involves design of compounds that could ligate that Cys. A second strategy involves the design of new molecules to target the catalytic serine of the enzyme. Here the challenge is not only to achieve high inhibition selectivity vs human AChE, but also to demonstrate toxicity to An. gambiae that carry the G119S resistance mutation of AgAChE. The advances made and challenges remaining will be presented. This review is part of the special issue "Insecticide Mode of Action: From Insect to Mammalian Toxicity"., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Widespread pyrethroid resistance has caused an urgent need to develop new insecticides for control of the malaria mosquito, Anopheles gambiae. Insecticide discovery efforts were directed towards the construction of bivalent inhibitors that occupy both the peripheral and catalytic sites of the mosquito acetylcholinesterase (AChE). It was hypothesized that this approach would yield a selective, high potency inhibitor that would also circumvent known catalytic site mutations (e.g. G119S) causing target site resistance. Accordingly, a series of bivalent phthalimide-pyrazole carbamates were prepared having an alkyl chain linker of varying length, along with other modifications. The most active compound was (1-(3-(1,3-dioxoisoindolin-2-yl)propyl)-1H-pyrazol-4-yl methylcarbamate, 8a), which has a chain length of three carbons, good mosquito anticholinesterase activity, and ca. 5-fold selectivity compared to human AChE. Moreover, this compound was toxic to mosquitoes by topical application (LD 50 = 63 ng/female) with only 6-fold cross resistance in the Akron strain of Anopheles gambiae that showed 50- to 60-fold resistance to conventional carbamate insecticides. However, contact lethality in the WHO paper assay was disappointing. The implications of these results for design of new mosquitocides are discussed.

Despite the troubling psychiatric side-effects it causes in some patients, mefloquine (MQ) has been used for malaria prophylaxis and therapy, due to its activity against all Plasmodium species, its ease of dosing, and its relative safety in children and pregnant women. Yet at present there is no consensus on the mechanism of antimalarial action of MQ. Two leading hypotheses for the mechanism of MQ are inhibition of heme crystallization and inhibition of host cell hemoglobin endocytosis. In this report we show that MQ is a potent and rapid inhibitor of amino acid efflux from intact parasitized erythrocytes, which is a measure of the in vivo rate of host hemoglobin endocytosis and catabolism. To further explore the mechanism of action of MQ, we have compared the effects of MQ and 18 non-piperidine analogs on amino acid efflux and parasite growth. Among these closely related compounds, an excellent correlation over nearly 4 log units is seen for 50% inhibition concentration (IC50) values for parasite growth and leucine efflux. These data and other observations are consistent with the hypothesis that the antimalarial action of these compounds derives from inhibition of hemoglobin endocytosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Coxiella burnetii is a highly infectious bacterium and potential agent of bioterrorism. However, it has not been studied as extensively as other biological agents, and very few of its proteins have been structurally characterized. To address this situation, we undertook a study of critical metabolic enzymes in C. burnetii that have great potential as drug targets. We used high-throughput techniques to produce novel crystal structures of 48 of these proteins. We selected one protein, C. burnetii dihydrofolate reductase (CbDHFR), for additional work to demonstrate the value of these structures for structure-based drug design. This enzyme's structure reveals a feature in the substrate binding groove that is different between CbDHFR and human dihydrofolate reductase (hDHFR). We then identified a compound by in silico screening that exploits this binding groove difference, and demonstrated that this compound inhibits CbDHFR with at least 25-fold greater potency than hDHFR. Since this binding groove feature is shared by many other prokaryotes, the compound identified could form the basis of a novel antibacterial agent effective against a broad spectrum of pathogenic bacteria., (© 2015 Wiley Periodicals, Inc.)

Malaria is a devastating disease in sub-Saharan Africa, and current vector control measures are threatened by emerging resistance mechanisms. With the goal of developing new, selective, resistance-breaking insecticides we explored α-fluorinated methyl ketones as reversible covalent inhibitors of Anopheles gambiae acetylcholinesterase (AgAChE). Trifluoromethyl ketones 5 demonstrated remarkable volatility in microtiter plate assays, but 5c,e-h exhibited potent (1-100 nM) inhibition of wild type (WT) AgAChE and weak inhibition of resistant mutant G119S mutant AgAChE. Fluoromethyl ketones 10c-i exhibited submicromolar to micromolar inhibition of WT AgAChE, but again only weakly inhibited G119S AgAChE. Interestingly, difluoromethyl ketone inhibitors 9c and 9g had single digit nanomolar inhibition of WT AgAChE, and 9g had excellent potency against G119S AgAChE. Approach to steady-state inhibition was quite slow, but after 23 h incubation an IC50 value of 25.1 ± 1.2 nM was measured. We attribute the slow, tight-binding G119S AgAChE inhibition of 9g to a balance of steric size and electrophilicity. However, toxicities of 5g, 9g, and 10g to adult A. gambiae in tarsal contact, fumigation, and injection assays were lower than expected based on WT AgAChE inhibition potency and volatility. Potential toxicity-limiting factors are discussed., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Insecticide resistance in the malaria mosquito Anopheles gambiae is well documented, and widespread agricultural use of pyrethroids may exacerbate development of resistance when pyrethroids are used in vector control. We have developed carbamate anticholinesterases that possess a high degree of An. gambiae:human selectivity for enzyme inhibition. The purpose of this study was to assess the spectrum of activity of these carbamates against other mosquitoes and agricultural pests., Results: Experimental carbamates were potent inhibitors of mosquito acetylcholinesterases, with IC50 values in the nanomolar range. Similar potencies were observed for Musca domestica and Drosophila melanogaster enzymes. Although meta-substituted carbamates were potent inhibitors, two ortho-substituted carbamates displayed poor enzyme inhibition (IC50 ≥ 10(-6)  M) in honey bee (Apis mellifera), Asian citrus psyllid (Diaphorina citri) and lepidopteran agricultural pests (Plutella xylostella and Ostrinia nubilalis). Enzyme inhibition results were confirmed by toxicity studies in caterpillars, where the new carbamates were 2-3-fold less toxic than propoxur and up to tenfold less active than bendiocarb, indicating little utility of these compounds for crop protection., Conclusion: The experimental carbamates were broadly active against mosquito species but not agricultural pests, which should mitigate selection for mosquito insecticide resistance by reducing agricultural uses of these compounds. © 2014 Society of Chemical Industry., (© 2014 Society of Chemical Industry.)

Insecticide resistance in the malaria vector, Anopheles gambiae, is a serious problem, epitomized by the multi-resistant Akron strain, originally isolated in the country of Benin. Here we report resistance in this strain to pyrethroids and DDT (13-fold to 35-fold compared to the susceptible G3 strain), but surprisingly little resistance to etofenprox, a compound sometimes described as a "pseudo-pyrethroid." There was also strong resistance to topically-applied commercial carbamates (45-fold to 81-fold), except for the oximes aldicarb and methomyl. Biochemical assays showed enhanced cytochrome P450 monooxygenase and carboxylesterase activity, but not that of glutathione-S-transferase. A series of substituted α,α,α,-trifluoroacetophenone oxime methylcarbamates were evaluated for enzyme inhibition potency and toxicity against G3 and Akron mosquitoes. The compound bearing an unsubstituted phenyl ring showed the greatest toxicity to mosquitoes of both strains. Low cross resistance in Akron was retained by all analogs in the series. Kinetic analysis of acetylcholinesterase activity and its inhibition by insecticides in the G3 strain showed inactivation rate constants greater than that of propoxur, and against Akron enzyme inactivation rate constants similar to that of aldicarb. However, inactivation rate constants against recombinant human AChE were essentially identical to that of the G3 strain. Thus, the acetophenone oxime carbamates described here, though potent insecticides that control resistant Akron mosquitoes, require further structural modification to attain acceptable selectivity and human safety., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Compounds that target isoprenoid biosynthesis in Plasmodium falciparum could be a welcome addition to malaria chemotherapy, since the methylerythritol phosphate (MEP) pathway used by the parasite is not present in humans. We previously reported that MMV008138 targets the apicoplast of P. falciparum and that its target in the MEP pathway differs from that of Fosmidomycin. In this Letter, we determine that the active stereoisomer of MMV008138 is 4a, which is (1R,3S)-configured. 2',4'-Disubstitution of the D ring was also found to be crucial for inhibition of the parasite growth. Limited variation of the C3-carboxylic acid substituent was carried out, and methylamide derivative 8a was found to be more potent than 4a; other amides, acylhydrazines, and esters were less potent. Finally, lead compounds 4a, 4e, 4f, 4h, 8a, and 8e did not inhibit growth of Escherichia coli, suggesting that protozoan-selective inhibition of the MEP pathway of P. falciparum can be achieved., (Copyright © 2015 Elsevier Ltd. All rights reserved.)