Select ß- and γ-branched 1-alkylpyrazol-4-yl methylcarbamates exhibit high selectivity for inhibition of Anopheles gambiae versus human acetylcholinesterase.

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 (AgAChE) 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 AgAChE 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 ß- 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 AgAChE 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 AgAChE, compared to that of W86 in hAChE. Compound 26a has reasonable contact toxicity to G3 strain An. gambiae (LC50 = 269 µg/mL) and low cross-resistance to Akron strain (LC50 = 948 µg/mL), which bears the G119S resistance mutation.

Bivalent Carbamates as Novel Control Agents of the Malaria Mosquito, Anopheles gambiae.

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 (LD50 = 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.

Difluoromethyl ketones: Potent inhibitors of wild type and carbamate-insensitive G119S mutant Anopheles gambiae acetylcholinesterase.

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.

3-Oxoisoxazole-2(3H)-carboxamides and isoxazol-3-yl carbamates: Resistance-breaking acetylcholinesterase inhibitors targeting the malaria mosquito, Anopheles gambiae.

To identify potential selective and resistance-breaking mosquitocides against the African malaria vector Anopheles gambiae, we investigated the acetylcholinesterase (AChE) inhibitory and mosquitocidal properties of isoxazol-3-yl dimethylcarbamates (15), and the corresponding 3-oxoisoxazole-2(3H)-dimethylcarboxamide isomers (14). In both series, compounds were found with excellent contact toxicity to wild-type susceptible (G3) strain and multiply resistant (Akron) strain mosquitoes that carry the G119S resistance mutation of AChE. Compounds possessing good to excellent toxicity to Akron strain mosquitoes inhibit the G119S mutant of An. gambiae AChE (AgAChE) with ki values at least 10- to 600-fold higher than that of propoxur, a compound that does not kill Akron mosquitoes at the highest concentration tested. On average, inactivation of WT AgAChE by dimethylcarboxamides 14 was 10-20 fold faster than that of the corresponding isoxazol-3-yl dimethylcarbamates 15. X-ray crystallography of dimethylcarboxamide 14d provided insight into that reactivity, a finding that may explain the inhibitory power of structurally-related inhibitors of hormone-sensitive lipase. Finally, human/An. gambiae AChE inhibition selectivities of these compounds were low, suggesting the need for additional structural modification.

Carbamate and pyrethroid resistance in the akron strain of Anopheles gambiae.

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.

Neurotoxicology of bis(n)-tacrines on Blattella germanica and Drosophila melanogaster acetylcholinesterase.

A series of bis(n)-tacrines were used as pharmacological probes of the acetylcholinesterase (AChE) catalytic and peripheral sites of Blattella germanica and Drosophila melanogaster, which express AChE-1 and AChE-2 isoforms, respectively. In general, the potency of bis(n)-tacrines was greater in D. melanogaster AChE (DmAChE) than in B. germanica AChE (BgAChE). The change in potency with tether length was high in DmAChE and low in BgAChE, associated with 90-fold and 5.2-fold maximal potency gain, respectively, compared to the tacrine monomer. The optimal tether length for Blattella was 8 carbons and for Drosophila was 10 carbons. The two species differed by only about twofold in their sensitivity to tacrine monomer, indicating that differential potency occurred among dimeric bis(n)-tacrines due to structural differences in the peripheral site. Multiple sequence alignment and in silico homology modeling suggest that aromatic residues of DmAChE confer higher affinity binding, and the lack of same at the BgAChE peripheral site may account, at least in part, to the greater overall sensitivity of DmAChE to bis(n)-tacrines, as reflected by in vitro assay data. Topical and injection assays in cockroaches found minimal toxicity of bis(n)-tacrines. Electrophysiological studies on D. melanogaster central nervous system showed that dimeric tacrines do not readily cross the blood brain barrier, explaining the observed nonlethality to insects. Although the bis(n)-tacrines were not good insecticide candidates, the information obtained in this study should aid in the design of selective bivalent ligands targeting insect, pests, and disease vectors.

Re-engineering aryl methylcarbamates to confer high selectivity for inhibition of Anopheles gambiae versus human acetylcholinesterase.

To identify potential human-safe insecticides against the malaria mosquito we undertook an investigation of the structure-activity relationship of aryl methylcarbamates inhibitors of acetylcholinesterase (AChE). Compounds bearing a ß-branched 2-alkoxy or 2-thioalkyl group were found to possess good selectivity for inhibition of Anopheles gambiae AChE over human AChE; up to 530-fold selectivity was achieved with carbamate 11d. A 3D QSAR model is presented that is reasonably consistent with log inhibition selectivity of 34 carbamates. Toxicity of these compounds to live Anopheles gambiae was demonstrated using both tarsal contact (filter paper) and topical application protocols.

Mosquito Acetylcholinesterase as a Target for Novel Phenyl-Substituted Carbamates.

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 AgAChE, but showed a smaller reduction in the inhibition of hAChE. A series of 4-carboxamido analogs of the parent compound gave reduced activity against AgAChE and generally showed more activity against hAChE than AgAChE. Replacement of the 3-t-buyl group with CF3 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.

Towards a species-selective acetylcholinesterase inhibitor to control the mosquito vector of malaria, Anopheles gambiae.

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.

Complexes of alkylene-linked tacrine dimers with Torpedo californica acetylcholinesterase: Binding of Bis5-tacrine produces a dramatic rearrangement in the active-site gorge.

The X-ray crystal structures were solved for complexes with Torpedo californica acetylcholinesterase of two bivalent tacrine derivative compounds in which the two tacrine rings were separated by 5- and 7-carbon spacers. The derivative with the 7-carbon spacer spans the length of the active-site gorge, making sandwich interactions with aromatic residues both in the catalytic anionic site (Trp84 and Phe330) at the bottom of the gorge and at the peripheral anionic site near its mouth (Tyr70 and Trp279). The derivative with the 5-carbon spacer interacts in a similar manner at the bottom of the gorge, but the shorter tether precludes a sandwich interaction at the peripheral anionic site. Although the upper tacrine group does interact with Trp279, it displaces the phenyl residue of Phe331, thus causing a major rearrangement in the Trp279-Ser291 loop. The ability of this inhibitor to induce large-scale structural changes in the active-site gorge of acetylcholinesterase has significant implications for structure-based drug design because such conformational changes in the target enzyme are difficult to predict and to model.

Aryl methylcarbamates: potency and selectivity towards wild-type and carbamate-insensitive (G119S) Anopheles gambiae acetylcholinesterase, and toxicity to G3 strain An. gambiae.

New carbamates that are highly selective for inhibition of Anopheles gambiae acetylcholinesterase (AChE) over the human enzyme might be useful in continuing efforts to limit malaria transmission. In this report we assessed 34 synthesized and commercial carbamates for their selectivity to inhibit the AChEs found in carbamate-susceptible (G3) and carbamate-resistant (Akron) An. gambiae, relative to human AChE. Excellent correspondence is seen between inhibition potencies measured with carbamate-susceptible mosquito homogenate and purified recombinant wild-type (WT) An. gambiae AChE (AgAChE). Similarly, excellent correspondence is seen between inhibition potencies measured with carbamate-resistant mosquito homogenate and purified recombinant G119S AgAChE, consistent with our earlier finding that the Akron strain carries the G119S mutation. Although high (100- to 500-fold) WT An. gambiae vs human selectivity is observed for several compounds, none of the carbamates tested potently inhibits the G119S mutant enzyme. Finally, we describe a predictive model for WT An. gambiae tarsal contact toxicity of the carbamates that relies on inhibition potency, molecular volume, and polar surface area.

Select small core structure carbamates exhibit high contact toxicity to "carbamate-resistant" strain malaria mosquitoes, Anopheles gambiae (Akron).

Acetylcholinesterase (AChE) is a proven target for control of the malaria mosquito (Anopheles gambiae). Unfortunately, a single amino acid mutation (G119S) in An. gambiae AChE-1 (AgAChE) confers resistance to the AChE inhibitors currently approved by the World Health Organization for indoor residual spraying. In this report, we describe several carbamate inhibitors that potently inhibit G119S AgAChE and that are contact-toxic to carbamate-resistant An. gambiae. PCR-RFLP analysis was used to confirm that carbamate-susceptible G3 and carbamate-resistant Akron strains of An. gambiae carry wild-type (WT) and G119S AChE, respectively. G119S AgAChE was expressed and purified for the first time, and was shown to have only 3% of the turnover number (k(cat)) of the WT enzyme. Twelve carbamates were then assayed for inhibition of these enzymes. High resistance ratios (>2,500-fold) were observed for carbamates bearing a benzene ring core, consistent with the carbamate-resistant phenotype of the G119S enzyme. Interestingly, resistance ratios for two oxime methylcarbamates, and for five pyrazol-4-yl methylcarbamates were found to be much lower (4- to 65-fold). The toxicities of these carbamates to live G3 and Akron strain An. gambiae were determined. As expected from the enzyme resistance ratios, carbamates bearing a benzene ring core showed low toxicity to Akron strain An. gambiae (LC(50)>5,000 µg/mL). However, one oxime methylcarbamate (aldicarb) and five pyrazol-4-yl methylcarbamates (4a-e) showed good to excellent toxicity to the Akron strain (LC(50) = 32-650 µg/mL). These results suggest that appropriately functionalized "small-core" carbamates could function as a resistance-breaking anticholinesterase insecticides against the malaria mosquito.

Crystal packing mediates enantioselective ligand recognition at the peripheral site of acetylcholinesterase.

Recently, alkylene-linked heterodimers of tacrine (1) and 5-amino-5,6,7,8-tetrahydroquinolinone (2, hupyridone) were shown to exhibit higher acetylcholinesterase (AChE) inhibition than either monomeric 1 or 2. Such inhibitors are potential drug candidates for ameliorating the cognitive decrements in early Alzheimer patients. In an attempt to understand the inhibition mechanism of one such dimer, (RS)-(+/-)-N-9-(1,2,3,4-tetrahydroacridinyl)-N'-5-[5,6,7,8-tetrahydro-2'(1'H)-quinolinonyl]-1,10-diaminodecane [(RS)-(+/-)-3] bisoxalate, the racemate was soaked in trigonal Torpedo californica AChE (TcAChE) crystals, and the X-ray structure of the resulting complex was solved to 2.30 A resolution. Its structure revealed the 1 unit bound to the "anionic" subsite of the active site, near the bottom of the active-site gorge, as seen for the 1/TcAChE complex. Interestingly, only the (R)-enantiomer of the 2 unit was seen in the peripheral "anionic" site (PAS) at the top of the gorge, and was hydrogen-bonded to the side chains of residues belonging to an adjacent, symmetry-related AChE molecule covering the gorge entrance. When the same racemate was soaked in orthorhombic crystals of TcAChE, in which the entrance to the gorge is more exposed, the crystal structure of the corresponding complex revealed no substantial enantiomeric selectivity. This observation suggests that the apparent enantiomeric selectivity of trigonal crystals of TcAChE for (R)-3 is mainly due to crystal packing, resulting in preferential binding of one enantiomeric inhibitor both to its "host" enzyme and to its neighbor in the asymmetric unit, rather than to steric constraints imposed by the geometry of the active-site gorge.

Catalytic asymmetric synthesis of protected tryptophan regioisomers.

Tryptophan 1 (Trp) is superior to all other naturally occurring peptide residues in its ability to bind cations (the cation-pi interaction). In an effort to expand the toolbox of Trp-like amino acids, in this note we report catalytic asymmetric syntheses of Trp regioisomers 2a-e, where the alanine unit is attached not to C-3 of indole but to C-2, C-4, C-5, C-6, or C-7. Excellent asymmetric induction is obtained in each case (generally >97% ee). Ab initio calculations suggest that the indole nuclei of 2a-e will bind Na(+) with the same affinity as that of Trp.

Acetylcholinesterase complexed with bivalent ligands related to huperzine a: experimental evidence for species-dependent protein-ligand complementarity.

Acetylcholinesterase (AChE) inhibitors improve the cognitive abilities of Alzheimer patients. (-)-Huperzine A [(-)-HupA], an alkaloid isolated from the club moss, Huperzia serrata, is one such inhibitor, but the search for more potent and selective drugs continues. Recently, alkylene-linked dimers of 5-amino-5,6,7,8-tetrahydroquinolinone (hupyridone, 1a), a fragment of HupA, were shown to serve as more potent inhibitors of AChE than (-)-HupA and monomeric 1a. We soaked two such dimers, (S,S)-(-)-bis(10)-hupyridone [(S,S)-(-)-2a] and (S,S)-(-)-bis(12)-hupyridone [(S,S)-(-)-2b] containing, respectively, 10 and 12 methylenes in the spacer, into trigonal TcAChE crystals, and solved the X-ray structures of the resulting complexes using the difference Fourier technique, both to 2.15 A resolution. The structures revealed one HupA-like 1a unit bound to the "anionic" subsite of the active-site, near the bottom of the active-site gorge, adjacent to Trp84, as seen for the TcAChE/(-)-HupA complex, and the second 1a unit near Trp279 in the "peripheral" anionic site at the top of the gorge, both bivalent molecules thus spanning the active-site gorge. The results confirm that the increased affinity of the dimeric HupA analogues for AChE is conferred by binding to the two "anionic" sites of the enzyme. Inhibition data show that (-)-2a binds to TcAChE approximately 6-7- and > 170-fold more tightly than (-)-2b and (-)-HupA, respectively. In contrast, previous data for rat AChE show that (-)-2b binds approximately 3- and approximately 2-fold more tightly than (-)-2a and (-)-HupA, respectively. Structural comparison of TcAChE with rat AChE, as represented by the closely related mouse AChE structure (1maa.pdb), reveals a narrower gorge for rat AChE, a perpendicular alignment of the Tyr337 ring to the gorge axis, and its conformational rigidity, as a result of hydrogen bonding between its hydroxyl group and that of Tyr341, relative to TcAChE Phe330. These structural differences in the active-site gorge explain the switch in inhibitory potency of (-)-2a and 2b and the larger dimer/(-)-HupA potency ratios observed for TcAChE relative to rat AChE. The results offer new insights into factors affecting protein-ligand complementarity within the gorge and should assist the further development of improved AChE inhibitors.