Structure-Activity Relationships Reveal Beneficial Selectivity Profiles of Inhibitors Targeting Acetylcholinesterase of Disease-Transmitting Mosquitoes.

Insecticide resistance jeopardizes the prevention of infectious diseases such as malaria and dengue fever by vector control of disease-transmitting mosquitoes. Effective new insecticidal compounds with minimal adverse effects on humans and the environment are therefore urgently needed. Here, we explore noncovalent inhibitors of the well-validated insecticidal target acetylcholinesterase (AChE) based on a 4-thiazolidinone scaffold. The 4-thiazolidinones inhibit AChE1 from the mosquitoes Anopheles gambiae and Aedes aegypti at low micromolar concentrations. Their selectivity depends primarily on the substitution pattern of the phenyl ring; halogen substituents have complex effects. The compounds also feature a pendant aliphatic amine that was important for activity; little variation of this group is tolerated. Molecular docking studies suggested that the tight selectivity profiles of these compounds are due to competition between two binding sites. Three 4-thiazolidinones tested for in vivo insecticidal activity had similar effects on disease-transmitting mosquitoes despite a 10-fold difference in their in vitro activity.

Broad-Spectrum Antidote Discovery by Untangling the Reactivation Mechanism of Nerve-Agent-Inhibited Acetylcholinesterase.

Reactivators are vital for the treatment of organophosphorus nerve agent (OPNA) intoxication but new alternatives are needed due to their limited clinical applicability. The toxicity of OPNAs stems from covalent inhibition of the essential enzyme acetylcholinesterase (AChE), which reactivators relieve via a chemical reaction with the inactivated enzyme. Here, we present new strategies and tools for developing reactivators. We discover suitable inhibitor scaffolds by using an activity-independent competition assay to study non-covalent interactions with OPNA-AChEs and transform these inhibitors into broad-spectrum reactivators. Moreover, we identify determinants of reactivation efficiency by analysing reactivation and pre-reactivation kinetics together with structural data. Our results show that new OPNA reactivators can be discovered rationally by exploiting detailed knowledge of the reactivation mechanism of OPNA-inhibited AChE.

Dual Reversible Coumarin Inhibitors Mutually Bound to Monoamine Oxidase B and Acetylcholinesterase Crystal Structures.

Multitarget directed ligands (MTDLs) represent a promising frontier in tackling the complexity of multifactorial pathologies. The synergistic inhibition of monoamine oxidase B (MAO B) and acetylcholinesterase (AChE) is believed to provide a potentiated effect in the treatment of Alzheimer's disease. Among previously reported micromolar or sub-micromolar coumarin-bearing dual inhibitors, compound 1 returned a tight-binding inhibition of MAO B (K i = 4.5 µM) and a +5.5 °C increase in the enzyme T m value. Indeed, the X-ray crystal structure revealed that binding of 1 produces unforeseen conformational changes at the MAO B entrance cavity. Interestingly, 1 showed great shape complementarity with the AChE enzymatic gorge, being deeply buried from the catalytic anionic subsite (CAS) to the peripheral anionic subsite (PAS) and causing significant structural changes in the active site. These findings provide structural templates for further development of dual MAO B and AChE inhibitors.

Characterization of toxicological effects of complex nano-sized metal particles using in vitro human cell and whole blood model systems.

Metal oxide fumes form at high temperatures, for instance, during welding or firing ammunition. Inhalation exposure to high levels of airborne metal oxide particles can cause metal fume fever, cardiovascular effects, and lung damage in humans, but the associated underlying pathological mechanisms are still not fully understood. Using human alveolar epithelial cells, vascular endothelial cells, and whole blood model systems, we aimed to elucidate the short-term effects of well-characterized metal particles emitted while firing pistol ammunition. Human lung epithelial cells exposed to gunshot smoke particles (0.1-50 µg/ml) produced reactive oxygen species (ROS) and pro-inflammatory cytokines (interleukin 8 (IL-8), granulocyte-macrophage colony-stimulating factor (GM-CSF)) that activate and recruit immune cells. Particles comprising high copper (Cu) and zinc (Zn) content activated human endothelial cells via a non-ROS-mediated mechanism that triggered immune activation (IL-8, GM-CSF), leukocyte adhesion to the endothelium (soluble intercellular adhesion molecule 1 (sICAM-1)), and secretion of regulators of the acute-phase protein synthesis (interleukin 6 (IL-6)). In human whole blood, metal oxides in gunshot smoke demonstrated intrinsic properties that activated platelets (release of soluble cluster of differentiation 40 ligand (sCD40L), platelet-derived growth factor B-chain homodimer(PDGF-BB), and vascular endothelial growth factor A (VEGF-A)) and blood coagulation and induced concomitant release of pro-inflammatory cytokines from blood leukocytes that further orchestrate thrombogenesis. The model systems applied provide useful tools for health risk assessment of particle exposures, but more studies are needed to further elucidate the mechanisms of metal fume fever and to evaluate the potential risk of long-term cardiovascular diseases.

In Situ Assembly of Choline Acetyltransferase Ligands by a Hydrothiolation Reaction Reveals Key Determinants for Inhibitor Design.

The potential drug target choline acetyltransferase (ChAT) catalyses the production of the neurotransmitter acetylcholine in cholinergic neurons, T-cells, and B-cells. Herein, we show that arylvinylpyridiniums (AVPs), the most widely studied class of ChAT inhibitors, act as substrate in an unusual coenzyme A-dependent hydrothiolation reaction. This in situ synthesis yields an adduct that is the actual enzyme inhibitor. The adduct is deeply buried in the active site tunnel of ChAT and interactions with a hydrophobic pocket near the choline binding site have major implications for the molecular recognition of inhibitors. Our findings clarify the inhibition mechanism of AVPs, establish a drug modality that exploits a target-catalysed reaction between exogenous and endogenous precursors, and provide new directions for the development of ChAT inhibitors with improved potency and bioactivity.

Physical Mechanisms Governing Substituent Effects on Arene-Arene Interactions in a Protein Milieu.

Arene-arene interactions play important roles in protein-ligand complex formation. Here, we investigate the characteristics of arene-arene interactions between small organic molecules and aromatic amino acids in protein interiors. The study is based on X-ray crystallographic data and quantum mechanical calculations using the enzyme acetylcholinesterase and selected inhibitory ligands as a model system. It is shown that the arene substituents of the inhibitors dictate the strength of the interaction and the geometry of the resulting complexes. Importantly, the calculated interaction energies correlate well with the measured inhibitor potency. Non-hydrogen substituents strengthened all interaction types in the protein milieu, in keeping with results for benzene dimer model systems. The interaction energies were dispersion-dominated, but substituents that induced local dipole moments increased the electrostatic contribution and thus yielded more strongly bound complexes. These findings provide fundamental insights into the physical mechanisms governing arene-arene interactions in the protein milieu and thus into molecular recognition between proteins and small molecules.

Noncovalent Inhibitors of Mosquito Acetylcholinesterase 1 with Resistance-Breaking Potency.

Resistance development in insects significantly threatens the important benefits obtained by insecticide usage in vector control of disease-transmitting insects. Discovery of new chemical entities with insecticidal activity is highly desired in order to develop new insecticide candidates. Here, we present the design, synthesis, and biological evaluation of phenoxyacetamide-based inhibitors of the essential enzyme acetylcholinesterase 1 (AChE1). AChE1 is a validated insecticide target to control mosquito vectors of, e.g., malaria, dengue, and Zika virus infections. The inhibitors combine a mosquito versus human AChE selectivity with a high potency also for the resistance-conferring mutation G122S; two properties that have proven challenging to combine in a single compound. Structure-activity relationship analyses and molecular dynamics simulations of inhibitor-protein complexes have provided insights that elucidate the molecular basis for these properties. We also show that the inhibitors demonstrate in vivo insecticidal activity on disease-transmitting mosquitoes. Our findings support the concept of noncovalent, selective, and resistance-breaking inhibitors of AChE1 as a promising approach for future insecticide development.

N-Aryl-N'-ethyleneaminothioureas effectively inhibit acetylcholinesterase 1 from disease-transmitting mosquitoes.

Vector control of disease-transmitting mosquitoes by insecticides has a central role in reducing the number of parasitic- and viral infection cases. The currently used insecticides are efficient, but safety concerns and the development of insecticide-resistant mosquito strains warrant the search for alternative compound classes for vector control. Here, we have designed and synthesized thiourea-based compounds as non-covalent inhibitors of acetylcholinesterase 1 (AChE1) from the mosquitoes Anopheles gambiae (An. gambiae) and Aedes aegypti (Ae. aegypti), as well as a naturally occurring resistant-conferring mutant. The N-aryl-N'-ethyleneaminothioureas proved to be inhibitors of AChE1; the most efficient one showed submicromolar potency. Importantly, the inhibitors exhibited selectivity over the human AChE (hAChE), which is desirable for new insecticides. The structure-activity relationship (SAR) analysis of the thioureas revealed that small changes in the chemical structure had a large effect on inhibition capacity. The thioureas showed to have different SAR when inhibiting AChE1 and hAChE, respectively, enabling an investigation of structure-selectivity relationships. Furthermore, insecticidal activity was demonstrated using adult and larvae An. gambiae and Ae. aegypti mosquitoes.

Divergent structure-activity relationships of structurally similar acetylcholinesterase inhibitors.

The molecular interactions between the enzyme acetylcholinesterase (AChE) and two compound classes consisting of N-[2-(diethylamino)ethyl]benzenesulfonamides and N-[2-(diethylamino)ethyl]benzenemethanesulfonamides have been investigated using organic synthesis, enzymatic assays, X-ray crystallography, and thermodynamic profiling. The inhibitors' aromatic properties were varied to establish structure-activity relationships (SAR) between the inhibitors and the peripheral anionic site (PAS) of AChE. The two structurally similar compound classes proved to have distinctly divergent SARs in terms of their inhibition capacity of AChE. Eight X-ray structures revealed that the two sets have different conformations in PAS. Furthermore, thermodynamic profiles of the binding between compounds and AChE revealed class-dependent differences of the entropy/enthalpy contributions to the free energy of binding. Further development of the entropy-favored compound class resulted in the synthesis of the most potent inhibitor and an extension beyond the established SARs. The divergent SARs will be utilized to develop reversible inhibitors of AChE into reactivators of nerve agent-inhibited AChE.

Structure of the C-terminal domain of the surface antigen SpaP from the caries pathogen Streptococcus mutans.

SpaP is a 1500-residue adhesin expressed on the surface of the caries-implicated bacterium Streptococcus mutans. SpaP is a member of the antigen I/II (AgI/II) family of proteins expressed by oral streptococci. These surface proteins are crucial for the incorporation of streptococci into dental plaque. The structure of the C-terminal domain of SpaP (residues 1136-1489) was solved and refined to 2.2 Šresolution with six molecules in the asymmetric unit. Similar to a related AgI/II structure, SpaP is stabilized by isopeptide bonds between lysine and asparagine side chains.

The changing faces of Streptococcus antigen I/II polypeptide family adhesins.

Streptococcus mutans antigen I/II (AgI/II) protein was one of the first cell wall-anchored adhesins identified in Gram-positive bacteria. It mediates attachment of S. mutans to tooth surfaces and has been a focus for immunization studies against dental caries. The AgI/II family polypeptides recognize salivary glycoproteins, and are also involved in biofilm formation, platelet aggregation, tissue invasion and immune modulation. The genes encoding AgI/II family polypeptides are found among Streptococcus species indigenous to the human mouth, as well as in Streptococcus pyogenes, S. agalactiae and S. suis. Evidence of functionalities for different regions of the AgI/II proteins has emerged. A sequence motif within the C-terminal portion of Streptococcus gordonii SspB (AgI/II) is bound by Porphyromonas gingivalis, thus promoting oral colonization by this anaerobic pathogen. The significance of other epitopes is now clearer following resolution of regional crystal structures. A new picture emerges of the central V (variable) region, predicted to contain a carbohydrate-binding trench, being projected from the cell surface by a stalk formed by an unusual association between an N-terminal alpha-helix and a C-terminal polyproline helix. This presentation mode might be important in determining functional conformations of other Gram-positive surface proteins that have adhesin domains flanked by alpha-helical and proline-rich regions.

Two intramolecular isopeptide bonds are identified in the crystal structure of the Streptococcus gordonii SspB C-terminal domain.

Streptococcus gordonii is a primary colonizer and is involved in the formation of dental plaque. This bacterium expresses several surface proteins. One of them is the adhesin SspB, which is a member of the Antigen I/II family of proteins. SspB is a large multi-domain protein that has interactions with surface molecules on other bacteria and on host cells, and is thus a key factor in the formation of biofilms. Here, we report the crystal structure of a truncated form of the SspB C-terminal domain, solved by single-wavelength anomalous dispersion to 1.5 A resolution. The structure represents the first of a C-terminal domain from a streptococcal Antigen I/II protein and is comprised of two structurally related beta-sandwich domains, C2 and C3, both with a Ca(2+) bound in equivalent positions. In each of the domains, a covalent isopeptide bond is observed between a lysine and an asparagine, a feature that is believed to be a common stabilization mechanism in Gram-positive surface proteins. S. gordonii biofilms contain attachment sites for the periodontal pathogen Porphyromonas gingivalis and the SspB C-terminal domain has been shown to have one such recognition motif, the SspB adherence region. The motif protrudes from the protein, and serves as a handle for attachment. The structure suggests several additional putative binding surfaces, and other binding clefts may be created when the full-length protein is folded.

Crystal structure of the variable domain of the Streptococcus gordonii surface protein SspB.

The Antigen I/II (AgI/II) family of proteins are cell wall anchored adhesins expressed on the surface of oral streptococci. The AgI/II proteins interact with molecules on other bacteria, on the surface of host cells, and with salivary proteins. Streptococcus gordonii is a commensal bacterium, and one of the primary colonizers that initiate the formation of the oral biofilm. S. gordonii expresses two AgI/II proteins, SspA and SspB that are closely related. One of the domains of SspB, called the variable (V-) domain, is significantly different from corresponding domains in SspA and all other AgI/II proteins. As a first step to elucidate the differences among these proteins, we have determined the crystal structure of the V-domain from S. gordonii SspB at 2.3 A resolution. The domain comprises a beta-supersandwich with a putative binding cleft stabilized by a metal ion. The overall structure of the SspB V-domain is similar to the previously reported V-domain of the Streptococcus mutans protein SpaP, despite their low sequence similarity. In spite of the conserved architecture of the binding cleft, the cavity is significantly smaller in SspB, which may provide clues about the difference in ligand specificity. We also verified that the metal in the binding cleft is a calcium ion, in concurrence with previous biological data. It was previously suggested that AgI/II V-domains are carbohydrate binding. However, we tested that hypothesis by screening the SspB V-domain for binding to over 400 glycoconjucates and found that the domain does not interact with any of the carbohydrates.

A crystallizable form of the Streptococcus gordonii surface antigen SspB C-domain obtained by limited proteolysis.

SspB is a 1500-residue adhesin expressed on the surface of the oral bacterium Streptococcus gordonii. Its interaction with other bacteria and host cells initiates the development of dental plaque. The full-length C-terminal domain of SspB was cloned, overexpressed in Escherichia coli and purified. However, the protein could not be crystallized. Limited proteolysis of the full-length C-domain identified a core fragment. The proteolysis product was cloned, expressed and purified. The protein was crystallized using the hanging-drop vapour-diffusion method. X-ray data were collected and processed to a maximum resolution of 2.1 A with 96.4% completeness. The crystals belonged to space group P2(1), with one molecule in the asymmetric unit, a solvent content of 33.7% and a corresponding Matthews coefficient of 1.85 A(3) Da(-1).