Influence of pH on indole-dependent heterodimeric interactions between Anopheles gambiae odorant-binding proteins OBP1 and OBP4.

Insect Odorant Binding Proteins (OBPs) constitute important components of their olfactory apparatus, as they are essential for odor recognition. OBPs undergo conformational changes upon pH change, altering their interactions with odorants. Moreover, they can form heterodimers with novel binding characteristics. Anopheles gambiae OBP1 and OBP4 were found capable of forming heterodimers possibly involved in the specific perception of the attractant indole. In order to understand how these OBPs interact in the presence of indole and to investigate the likelihood of a pH-dependent heterodimerization mechanism, the crystal structures of OBP4 at pH 4.6 and 8.5 were determined. Structural comparison to each other and with the OBP4-indole complex (3Q8I, pH 6.85) revealed a flexible N-terminus and conformational changes in the α4-loop-α5 region at acidic pH. Fluorescence competition assays showed a weak binding of indole to OBP4 that becomes further impaired at acidic pH. Additional Molecular Dynamic and Differential Scanning Calorimetry studies displayed that the influence of pH on OBP4 stability is significant compared to the modest effect of indole. Furthermore, OBP1-OBP4 heterodimeric models were generated at pH 4.5, 6.5, and 8.5, and compared concerning their interface energy and cross-correlated motions in the absence and presence of indole. The results indicate that the increase in pH may induce the stabilization of OBP4 by increasing its helicity, thereby enabling indole binding at neutral pH that further stabilizes the protein and possibly promotes the creation of a binding site for OBP1. A decrease in interface stability and loss of correlated motions upon transition to acidic pH may provoke the heterodimeric dissociation allowing indole release. Finally, we propose a potential OBP1-OBP4 heterodimer formation/disruption mechanism induced by pH change and indole binding.

2D finger-printing and molecular docking studies identified potent mosquito repellents targeting odorant binding protein 1.

Personal protection measures against the mosquitoes like the use of repellents constitute valuable tools in the effort to prevent the transmission of vector-borne diseases. Therefore, the discovery of novel repellent molecules which will be effective at lower concentrations and provide a longer duration of protection remains an urgent need. Mosquito Odorant-Binding Proteins (OBPs) involved in the initial steps of the olfactory signal transduction cascade have been recognized not only as passive carriers of odors and pheromones but also as the first molecular filter to discriminate semiochemicals, hence serving as molecular targets for the design of novel pest control agents. Among the three-dimensional structures of mosquito OBPs solved in the last decades, the OBP1 complexes with known repellents have been widely used as reference structures in docking analysis and molecular dynamics simulation studies for the structure-based discovery of new molecules with repellent activity. Herein, ten compounds known to be active against mosquitoes and/or displaying a binding affinity for Anopheles gambiae AgamOBP1 were used as queries in an in silico screening of over 96 million chemical samples in order to detect molecules with structural similarity. Further filtering of the acquired hits on the basis of toxicity, vapor pressure, and commercial availability resulted in 120 unique molecules that were subjected to molecular docking studies against OBP1. For seventeen potential OBP1-binders, the free energy of binding (FEB) and mode of interaction with the protein were further estimated by molecular docking simulations leading to the selection of eight molecules exhibiting the highest similarity with their parental compounds and favorable energy values. The in vitro determination of their binding affinity to AgamOBP1 and the evaluation of their repellent activity against female Aedes albopictus mosquitoes revealed that our combined ligand similarity screening and OBP1 structure-based molecular docking successfully detected three molecules with enhanced repellent properties. A novel DEET-like repellent with lower volatility (8.55 × 10-4 mmHg) but a higher binding affinity for OBP1 than DEET (1.35 × 10-3 mmHg). A highly active repellent molecule that is predicted to bind to the secondary Icaridin (sIC)-binding site of OBP1 with higher affinity than to the DEET-site and, therefore, represents a new scaffold to be exploited for the discovery of binders targeting multiple OBP sites. Finally, a third potent repellent exhibiting a high degree of volatility was found to be a strong DEET-site binder of OBP1 that could be used in slow-release formulations.

The structure of AgamOBP5 in complex with the natural insect repellents Carvacrol and Thymol: Crystallographic, fluorescence and thermodynamic binding studies.

Among several proteins participating in the olfactory perception process of insects, Odorant Binding Proteins (OBPs) are today considered valid targets for the discovery of compounds that interfere with their host-detection behavior. The 3D structures of Anopheles gambiae mosquito AgamOBP1 in complex with the known synthetic repellents DEET and Icaridin have provided valuable information on the structural characteristics that govern their selective binding. However, no structure of a plant-derived repellent bound to an OBP has been available until now. Herein, we present the novel three-dimensional crystal structures of AgamOBP5 in complex with two natural phenolic monoterpenoid repellents, Carvacrol and Thymol, and the MPD molecule. Structural analysis revealed that both monoterpenoids occupy a binding site (Site-1) by adopting two alternative conformations. An additional Carvacrol was also bound to a secondary site (Site-2) near the central cavity entrance. A protein-ligand hydrogen-bond network supplemented by van der Waals interactions spans the entire binding cavity, bridging α4, α6, and α3 helices and stabilizing the overall structure. Fluorescence competition and Differential Scanning Calorimetry experiments verified the presence of two binding sites and the stabilization effect on AgamOBP5. While Carvacrol and Thymol bind to Site-1 with equal affinity in the submicromolar range, they exhibit a significantly lower and distinct binding capacity for Site-2 with Kd's of ~7 µΜ and ~18 µΜ, respectively. Finally, a comparison of AgamOBP5 complexes with the AgamOBP4-Indole structure revealed that variations of ligand-interacting aminoacids such as A109T, I72M, A112L, and A105T cause two structurally similar and homologous proteins to display different binding specificities.

A Combined Computational Methodology for the Discovery of Hit Compounds with Putative Insect Repellency Properties.

Mosquitoes and other hematophagous arthropods, the primary vectors of multiple parasites and viruses, are responsible for the transmission of serious diseases to humans. Nowadays, the interest is focused on the development of novel repellents to the existing ones with advanced properties. The present study attempts the discovery of novel hit compounds which may evolve as insect repellents using a combined computational methodology targeting the Odorant Binding Protein 1 (OBP1). The in silico results indicated two compounds, namely coniferyl alcohol and 1,2-diphenyl-2-propanol, which were further evaluated (a) in vitro for their binding affinity to AgamOBP1 and (b) in vivo using dose-dependent repellence tests against the aggressive-day biting Aedes albopictus. The combination of in vitro and in vivo results pointed that coniferyl alcohol and 1,2-diphenyl-2-propanol exhibited high binding affinity over OBP1 with 69.4 and 84.7 nM, respectively as well as efficient repellent activity. Compounds were also tested for their dose-dependent repellency activity in vivo against Aedes albopictus. Overall, the selected compounds can serve as scaffolds for the development of novel repellents.

Curcumin Derivatives as Potential Mosquito Larvicidal Agents against Two Mosquito Vectors, Culex pipiens and Aedes albopictus.

Vector-borne diseases have appeared or re-emerged in many Southern Europe countries making the transmission of infectious diseases by mosquitoes (vectors) one of the greatest worldwide health threats. Larvicides have been used extensively for the control of Aedes (Stegomyia) albopictus (Skuse, 1895) (Diptera: Culicidae) and Culex pipiens Linnaeus, 1758 (Diptera: Culicidae) mosquitoes in urban and semi-urban environments, causing the increasing resistance of mosquitoes to commercial insecticides. In this study, 27 curcuminoids and monocarbonyl curcumin derivatives were synthesised and evaluated as potential larvicidal agents against Cx. pipiens and Ae. albopictus. Most of the compounds were more effective against larvae of both mosquito species. Four of the tested compounds, curcumin, demethoxycurcumin, curcumin-BF2 complex and a monocarbonyl tetramethoxy curcumin derivative exhibited high activity against both species. In Cx. pipiens the recorded LC50 values were 6.0, 9.4, 5.0 and 32.5 ppm, respectively, whereas in Ae. albopictus they exhibited LC50 values of 9.2, 36.0, 5.5 and 23.6 ppm, respectively. No conclusive structure activity relationship was evident from the results and the variety of descriptors values generated in silico provided some insight to this end.

The architecture of hydrogen and sulfur σ-hole interactions explain differences in the inhibitory potency of C-ß-d-glucopyranosyl thiazoles, imidazoles and an N-ß-d glucopyranosyl tetrazole for human liver glycogen phosphorylase and offer new insights to structure-based design.

C-Glucopyranosyl imidazoles, thiazoles, and an N-glucopyranosyl tetrazole were assessed in vitro and ex vivo for their inhibitory efficiency against isoforms of glycogen phosphorylase (GP; a validated pharmacological target for the development of anti-hyperglycaemic agents). Imidazoles proved to be more potent inhibitors than the corresponding thiazoles or the tetrazole. The most potent derivative has a 2-naphthyl substituent, a Ki value of 3.2 µM for hepatic glycogen phosphorylase, displaying also 60% inhibition of GP activity in HepG2 cells, compared to control vehicle treated cells, at 100 µM. X-Ray crystallography studies of the protein - inhibitor complexes revealed the importance of the architecture of inhibitor associated hydrogen bonds or sulfur σ-hole bond interactions to Asn284 OD1, offering new insights to structure-based design efforts. Moreover, while the 2-glucopyranosyl-tetrazole seems to bind differently from the corresponding 1,2,3-triazole compound, the two inhibitors are equipotent.

Glucopyranosylidene-spiro-imidazolinones, a New Ring System: Synthesis and Evaluation as Glycogen Phosphorylase Inhibitors by Enzyme Kinetics and X-ray Crystallography.

Epimeric series of aryl-substituted glucopyranosylidene-spiro-imidazolinones, an unprecedented new ring system, were synthesized from the corresponding Schiff bases of O-perbenzoylated (gluculopyranosylamine)onamides by intramolecular ring closure of the aldimine moieties with the carboxamide group elicited by N-bromosuccinimide in pyridine. Test compounds were obtained by Zemplén O-debenzoylation. Stereochemistry and ring tautomers of the new compounds were investigated by NMR, time-dependent density functional theory (TDDFT)-electronic circular dichroism, and DFT-NMR methods. Kinetic studies with rabbit muscle and human liver glycogen phosphorylases showed that the (R)-imidazolinones were 14-216 times more potent than the (S) epimers. The 2-naphthyl-substituted (R)-imidazolinone was the best inhibitor of the human enzyme (Ki 1.7 µM) and also acted on HepG2 cells (IC50 177 µM). X-ray crystallography revealed that only the (R) epimers bound in the crystal. Their inhibitory efficacy is based on the hydrogen-bonding interactions of the carbonyl oxygen and the NH of the imidazolinone ring.

High Consistency of Structure-Based Design and X-Ray Crystallography: Design, Synthesis, Kinetic Evaluation and Crystallographic Binding Mode Determination of Biphenyl-N-acyl-ß-d-Glucopyranosylamines as Glycogen Phosphorylase Inhibitors.

Structure-based design and synthesis of two biphenyl-N-acyl-ß-d-glucopyranosylamine derivatives as well as their assessment as inhibitors of human liver glycogen phosphorylase (hlGPa, a pharmaceutical target for type 2 diabetes) is presented. X-ray crystallography revealed the importance of structural water molecules and that the inhibitory efficacy correlates with the degree of disturbance caused by the inhibitor binding to a loop crucial for the catalytic mechanism. The in silico-derived models of the binding mode generated during the design process corresponded very well with the crystallographic data.

Affinity Crystallography Reveals the Bioactive Compounds of Industrial Juicing Byproducts of Punica granatum for Glycogen Phosphorylase.

BACKGROUND: Glycogen phosphorylase (GP) is a pharmaceutical target for the discovery of new antihyperglycaemic agents. Punica granatum is a well-known plant for its potent antioxidant and antimicrobial activities but so far has not been examined for antihyperglycaemic activity. OBJECTIVE: The aim was to examine the inhibitory potency of eighteen polyphenolic extracts obtained from Punica granatum fruits and industrial juicing byproducts against GP and discover their most bioactive ingredients. METHOD: Kinetic experiments were conducted to measure the IC50 values of the extracts while affinity crystallography was used to identify the most bioactive ingredient. The inhibitory effect of one of the polyphenolic extracts was also verified ex vivo, in HepG2 cells. RESULTS: All extracts exhibited significant in vitro inhibitory potency (IC50 values in the range of low µg/mL). Affinity crystallography revealed that the most bioactive ingredients of the extracts were chlorogenic and ellagic acids, found bound in the active and the inhibitor site of GP, respectively.While ellagic acid is an established GP inhibitor, the inhibition of chlorogenic acid is reported for the first time. Kinetic analysis indicated that chlorogenic acid is an inhibitor with Ki=2.5 x 10-3Mthat acts synergistically with ellagic acid. CONCLUSION: Our study provides the first evidence for a potential antidiabetic usage of Punica granatum extracts as antidiabetic food supplements. Although, more in vivo studies have to be performed before these extracts reach the stage of antidiabetic food supplements, our study provides a first positive step towards this process.

The ammonium sulfate inhibition of human angiogenin.

In this study, we investigate the inhibition of human angiogenin by ammonium sulfate. The inhibitory potency of ammonium sulfate for human angiogenin (IC50 = 123.5 ± 14.9 mm) is comparable to that previously reported for RNase A (119.0 ± 6.5 mm) and RNase 2 (95.7 ± 9.3 mm). However, analysis of two X-ray crystal structures of human angiogenin in complex with sulfate anions (in acidic and basic pH environments, respectively) indicates an entirely distinct mechanism of inhibition. While ammonium sulfate inhibits the ribonucleolytic activity of RNase A and RNase 2 by binding to the active site of these enzymes, sulfate anions bind only to peripheral substrate anion-binding subsites of human angiogenin, and not to the active site.