The Adipokinetic Hormone (AKH) and the Adipokinetic Hormone/Corazonin-Related Peptide (ACP) Signalling Systems of the Yellow Fever Mosquito Aedes aegypti: Chemical Models of Binding.

Neuropeptides are the main regulators of physiological, developmental, and behavioural processes in insects. Three insect neuropeptide systems, the adipokinetic hormone (AKH), corazonin (Crz), and adipokinetic hormone/corazonin-related peptide (ACP), and their cognate receptors, are related to the vertebrate gonadotropin (GnRH) system and form the GnRH superfamily of peptides. In the current study, the two signalling systems, AKH and ACP, of the yellow fever mosquito, Aedes aegypti, were comparatively investigated with respect to ligand binding to their respective receptors. To achieve this, the solution structure of the hormones was determined by nuclear magnetic resonance distance restraint methodology. Atomic-scale models of the two G protein-coupled receptors were constructed with the help of homology modelling. Thereafter, the binding sites of the receptors were identified by blind docking of the ligands to the receptors, and models were derived for each hormone system showing how the ligands are bound to their receptors. Lastly, the two models were validated by comparing the computational results with experimentally derived data available from the literature. This mostly resulted in an acceptable agreement, proving the models to be largely correct and usable. The identification of an antagonist versus a true agonist may, however, require additional testing. The computational data also explains the exclusivity of the two systems that bind only the cognate ligand. This study forms the basis for further drug discovery studies.

In Silico Screening for Pesticide Candidates against the Desert Locust Schistocerca gregaria.

Adipokinetic hormone (AKH) is one of the most important metabolic neuropeptides in insects, with actions similar to glucagon in vertebrates. AKH regulates carbohydrate and fat metabolism by mobilizing trehalose and diacylglycerol into circulation from glycogen and triacylglycerol stores, respectively, in the fat body. The short peptide (8 to 10 amino acids long) exerts its function by binding to a rhodopsin-like G protein-coupled receptor located in the cell membrane of the fat body. The AKH receptor (AKHR) is, thus, a potential target for the development of novel specific (peptide) mimetics to control pest insects, such as locusts, which are feared for their prolific breeding, swarm-forming behavior and voracious appetite. Previously, we proposed a model of the interaction between the three endogenous AKHs of the desert locust, Schistocerca gregaria, and the cognate AKHR (Jackson et al., Peer J. 7, e7514, 2019). In the current study we have performed in silico screening of two databases (NCI Open 2012 library and Zinc20) to identify compounds which may fit the endogenous Schgr-AKH-II binding site on the AKHR of S. gregaria. In all, 354 compounds were found to fit the binding site with glide scores < −8. Using the glide scores and binding energies, 7 docked compounds were selected for molecular dynamic simulation in a phosphatidylcholine membrane. Of these 7 compounds, 4 had binding energies which would allow them to compete with Schgr-AKH-II for the receptor binding site and so are proposed as agonistic ligand candidates. One of the ligands, ZINC000257251537, was tested in a homospecific in vivo biological assay and found to have significant antagonistic activity.

Insights into the Activation of a Crustacean G Protein-Coupled Receptor: Evaluation of the Red Pigment-Concentrating Hormone Receptor of the Water Flea Daphnia pulex (Dappu-RPCH R).

The validation of a previously developed model of the interaction between the red pigment-concentrating hormone of Daphnia pulex and its cognate receptor (Jackson et al., IJBM 106, 969-978, 2018) was undertaken. Single amino acid replacements, noticeably an Ala scan, of the ligand, Dappu-RPCH, were docked to the receptor, and the binding energies calculated and compared to the one with Dappu-RPCH. As a second step, the same molecules were docked using molecular dynamics (MD) in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane. Changes in binding energy were compared to previous results on in vitro receptor activation (Marco et al., Sci. Rep. 7, 6851, 2017). Residue scanning and MD simulations both gave comparable results for binding energy. For most mutants, there was a good inverse correlation between in vitro activity and binding. There were, however, exceptions; for example: [Ala4]Dappu-RPCH bound as tightly as the cognate ligand but had little activity. This seeming discrepancy was explained when the MD data were analyzed in detail, showing that, although [Ala4]Dappu-RPCH had multiple interactions with the receptor accounting for the high binding energy, the interacting residues of the receptor were quite different to those of Dappu-RPCH. The MD calculations show clearly that the strong binding affinity of the ligand to the receptor is not sufficient for activation. Interaction of the binding of the ligand to two residues of the receptor, Ser 155 and Gln 237, is also essential. A comparison of our computational results with the experimental results of Marco et al. and comparison with the extensive data on GnRH supports the validity of our Dappu-RPCH R model.

Conformational analysis of a cyclic AKH neuropeptide analog that elicits selective activity on locust versus honeybee receptor.

Neuropeptides belonging to the adipokinetic hormone (AKH) family elicit metabolic effects as their main function in insects, by mobilizing trehalose, diacylgycerol, or proline, which are released from the fat body into the hemolymph as energy sources for muscle contraction required for energy-intensive processes, such as locomotion. One of the AKHs produced in locusts is a decapeptide, Locmi-AKH-I (pELNFTPNWGT-NH2). A head-to-tail cyclic, octapeptide analog of Locmi-AKH-I, cycloAKH (cyclo[LNFTPNWG]) was synthesized to severely restrict the conformational freedom of the AKH structure. In vitro, cycloAKH selectively retains full efficacy on a pest insect (desert locust) AKH receptor, while showing little or no activation of the AKH receptor of a beneficial insect (honeybee). Molecular dynamic analysis incorporating NMR data indicate that cycloAKH preferentially adopts a type II ß-turn under micelle conditions, whereas its linear counterpart and natural AKH adopts a type VI ß-turn under similar conditions. CycloAKH, linear LNFTPNWG-NH2, and Locmi-AKH-I feature the same binding site during docking simulations with the desert locust AKH receptor (Schgr-AKHR), but differ in the details of the ligand/receptor interactions. However, cycloAKH failed to enter the binding pocket of the honeybee receptor 3D model during docking simulations. Since the locust AKH receptor has a greater tolerance than the honeybee receptor for the cyclic conformational constraint in vitro receptor assays, it could suggest a greater tolerance for a shift in the direction of the type II ß turn exhibited by cycloAKH from the type VI ß turn of the linear octapeptide and the native locust decapeptide AKH. Selectivity in biostable mimetic analogs could potentially be enhanced by incorporating conformational constraints that emphasize this shift. Biostable mimetic analogs of AKH offer the potential of selectively disrupting AKH-regulated processes, leading to novel, environmentally benign control strategies for pest insect populations.

Successful urinary discrimination between calcium oxalate kidney stone patients and healthy subjects using 1 H NMR spectroscopy: Suggestion of a possible link to protein content.

Stone formation in the urinary tract is a multifactorial world-wide disease afflicting between 8 and 20% of population groups in different geographical locations. Discrimination between stone formers and healthy persons on the basis of urine composition remains a crucial goal among researchers. Since 1 H NMR is able to monitor the metabolic function of the kidney we applied it to the urine of 60 stone formers (34 females, 26 males) and 38 healthy persons (14 females, 24 males). Spectra were normalized relative to an internal standard and integrated over 37 consecutive regions. The resulting data were subjected to principal component and canonical discriminant analysis. Excellent discrimination between patient and controls for both genders was achieved, with all the data falling within the 95% confidence interval. The most important variables allowing for this inter-group separation correspond to those associated with protein signals. We therefore speculate that the discrimination between patients and controls may be due to the presence or absence of macromolecular stone promoters and/or inhibitors. This supports numerous in vitro studies demonstrating that urinary macromolecules play significant roles in stone formation and prevention. Our finding that 1 H NMR analysis of urine differentiates between stone formers and healthy persons represents an important breakthrough for rapid screening of individuals who are at risk for this disease.

The adipokinetic hormones and their cognate receptor from the desert locust, Schistocerca gregaria: solution structure of endogenous peptides and models of their binding to the receptor.

BACKGROUND: Neuropeptides exert their activity through binding to G protein-coupled receptors (GPCRs). GPCRs are well-known drug targets in the pharmaceutical industry and are currently discussed as targets to control pest insects. Here, we investigate the neuropeptide adipokinetic hormone (AKH) system of the desert locust Schistocerca gregaria. The desert locust is known for its high reproduction, and for forming devastating swarms consisting of billions of individual insects. It is also known that S. gregaria produces three different AKHs as ligands but has only one AKH receptor (AKHR). The AKH system is known to be essential for metabolic regulation, which is necessary for reproduction and flight activity. METHODS: Nuclear magnetic resonance techniques (NMR) in a dodecylphosphocholin (DPC) micelle solution were used to determine the structure of the three AKHs. The primary sequence of the S. gregaria AKHR was used to construct a 3D molecular model. Next, the three AKHs were individually docked to the receptor, and dynamic simulation of the whole ligand-receptor complex in a model membrane was performed. RESULTS: Although the three endogenous AKHs of S. gregaria have quite different amino acids sequences and chain length (two octa- and one decapeptide), NMR experiments assigned a turn structure in DPC micelle solution for all. The GPCR-ModSim program identified human kappa opioid receptor to be the best template after which the S. gregaria AKHR was modeled. All three AKHs were found to have the same binding site on this receptor, interact with similar residues of the receptor and have comparable binding constants. Molecular switches were also identified; the movement of the receptor could be visually shown when ligands (AKHs) were docked and the receptor was activated. CONCLUSIONS: The study proposes a model of binding of the three endogenous ligands to the one existing AKHR in the desert locust and paves the way to use such a model for the design of peptide analogs and finally, peptide mimetics, in the search for novel species-specific insecticides based on receptor-ligand interaction.

Interaction of the red pigment-concentrating hormone of the crustacean Daphnia pulex, with its cognate receptor, Dappu-RPCHR: A nuclear magnetic resonance and modeling study.

The primary sequence of the red pigment-concentrating hormone (RPCH) receptor of the water flea, Daphnia pulex, was used in homology modeling to construct the first 3D model of a crustacean G-protein coupled receptor, Dappu-RPCHR. This receptor was found to belong to the class A subfamily of GPCRs with a disulfide bridge between Cys72 and Cys150 and an ionic lock between Arg97 and Thr224 and Thr220. NMR restrained molecular dynamics was used to determine the structure of an agonist, Dappu-RPCH, in a membrane-mimicking environment. The agonist was found to be flexible but has two main conformations in solution, both having ß-turns. Docking of the predominant structure was used to find a binding pocket on the receptor. The pocket's spatial location was similar to that of the AKH receptor of Anopheles gambiae. The binding affinity was -69kcalmol-1 with the N-terminus of Dappu-RPCH inserted between helices 4 and 6, and the C-terminus interacting with extra-cellular loop, ECL2. Upon binding, H-bonding to the peptide may activate the receptor. This development of the first Dappu-RPCH/Dappu-RPCHR model could be useful for understanding ligand-receptor interactions in crustaceans.

Data for the homology modelling of the red pigment-concentrating hormone receptor (Dappu-RPCHR) of the crustacean Daphnia pulex, and docking of its cognate agonist (Dappu-RPCH).

The data presented in this article are related to the publication "Interaction of the red pigment-concentrating hormone of the crustacean Daphnia pulex, with its cognate receptor, Dappu-RPCHR: A nuclear magnetic resonance and modeling study" (Jackson et al., 2017) [1]. This article contains the data for homology modeling of the red pigment-concentrating hormone (RPCH) receptor of the water flea, Daphnia pulex (Dappu-RPCHR), which was constructed from its primary sequence. This is the first 3D model of a crustacean G-protein coupled receptor. Docking of the agonist, pGlu-Val-Asn-Phe-Ser-Thr-Ser-Trp amide (Dappu-RPCH), was used to find a binding pocket on the receptor and compared to the binding pocket of the adipokinetic hormone (AKH) receptor from the malaria mosquito. Data for the receptor, with and without loop refinement, together with the docked agonist, are presented.

Chitosan encapsulation of essential oil "cocktails" with well-defined binary Zn(II)-Schiff base species targeting antibacterial medicinal nanotechnology.

The advent of biodegradable nanomaterials with enhanced antibacterial activity stands as a challenge to the global research community. In an attempt to pursue the development of novel antibacterial medicinal nanotechnology, we herein a) synthesized ionic-gelated chitosan nanoparticles, b) compared and evaluated the antibacterial activity of essential oils extracted from nine different herbs (Greek origin) and their combinations with a well-defined antibacterial Zn(II)-Schiff base compound, and c) encapsulated the most effective hybrid combination of Zn(II)-essential oils inside the chitosan matrix, thereby targeting well-formulated nanoparticles of distinct biological impact. The empty and loaded chitosan nanoparticles were physicochemically characterized by FT-IR, Thermogravimetric Analysis (TGA), Scanning Electron Microscopy (SEM), with the entrapment and drug release studies being conducted through UV-Visible and atomic absorption techniques. The antimicrobial properties of the novel hybrid materials were demonstrated against Gram positive (S. aureus, B. subtilis, and B. cereus) and Gram negative (E. coli and X. campestris) bacteria using modified agar diffusion methods. The collective physicochemical profile of the hybrid Zn(II)-essential oil cocktails, formulated so as to achieve optimal activity when loaded to chitosan nanoparticles, signifies the importance of design in the development of efficient nanomedicinal pharmaceuticals a) based on both natural products and biogenic metal ionic cofactors, and b) targeting bacterial infections and drug resistance.

Solution equilibria and the X-ray structure of Cu(ii) complexation with 3-amino-N-(pyridin-2-ylmethyl)propanamide, a pseudo-mimic of human serum albumin.

Copper complexes have anti-inflammatory activity in the treatment of inflammation associated with rheumatoid arthritis (RA). The preferred route of administration is through the skin, so the rate of dermal absorption and bioavailability of copper is important. Based on previous studies, 3-amino-N-(pyridin-2-ylmethyl)-propanamide, [H(56)NH2], was designed as a potential chelator of copper. The stability constant measurements revealed that MLH-1 is the most stable species at the physiological pH of 7.4. The X-ray crystal structure of this species was solved and copper was found in a rectangular pyramidal geometry. The ligand occupied three coordination sites while bridging chloride linked copper ions together in a chain. The ligand bound to the metal ion through the pyridyl nitrogen, the amide nitrogen and the terminal amino group. Spectroscopic studies confirmed that this structure persisted in aqueous solution. Octanol/water partition coefficients and Franz cell permeation studies showed that [H(56)NH2] is able to promote the dermal absorption of Cu(ii).

Capsular polysaccharide conformations in pneumococcal serotypes 19F and 19A.

Streptococcus pneumoniae is a significant pathogen in children. Although the PCV7 pneumococcal conjugate vaccine has reduced pneumococcal disease, non-vaccine serotype 19A infection has increased, despite expectations of cross-protection from vaccine serotype 19F. Serotype 19A is included in the new PCV13 vaccine, but not in PCV10. In the solution simulations of 19F and 19A oligosaccharide chains reported here, both polysaccharides form unstructured random coils, with inflexible repeat units linked by mobile phosphodiester linkages. However, there are clear conformational differences. In the 19F repeat unit, the rhamnose residue is nearly orthogonal to the other residues, whereas 19A has residues in similar orientations. This finding is corroborated by key inter-residue distances calculated from NMR NOESY experiments. Further, 19F is predominantly in extended conformations, whereas 19A exhibits a high prevalence of tight hairpin bends. These conformational differences may account for a lack of antibody cross-protection between serotypes 19F and 19A.

Quercetin encapsulation in modified silica nanoparticles: potential use against Cu(II)-induced oxidative stress in neurodegeneration.

Neurodegenerative diseases entail deeply complex processes, intimately associated with progressive brain damage reflecting cellular demise. Biochemical reactivity linked to such processes in Alzheimer's disease involves, among others, metal-induced oxidative stress contributing to neuronal cell death. Prominent among redox active metals inducing oxidative stress is Cu(II). Poised to develop molecular technology counteracting oxidative stress, efforts were launched to prepare bioactive hybrid nanoparticles, capable of working as host-carriers of potent antioxidants, such as the natural flavonoid quercetin. Employing synthetic protocols consistent with the assembly of silica nanoparticles, PEGylated and CTAB-modified materials were synthesized. Subsequent concentration-dependent loading of quercetin led to well-defined molecular carriers, the antioxidant efficiency of which was determined through drug release studies using UV-visible spectroscopy. The physicochemical characterization (elemental analysis, particle size, z-potential, FT-IR, thermogravimetric analysis, scanning electron microscopy) of the empty and loaded silica nanoparticles led to the formulation of optimized material linked to the delivery of the encapsulated antioxidant to primary rat hippocampal cultures under oxidative stress. Entrapment and drug release studies showed a) the competence of hybrid nanoparticles as far as the loading capacity in quercetin (concentration dependence), b) congruence with the physicochemical features determined, and c) the release profile of the nanoparticle load under oxidative stress in neuronal cultures. The bio-activity profile of quercetin nanoparticles in a neurodegenerative environment brought on by Cu(II) a) denotes the improved specificity of antioxidant reactivity counteracting oxidative stress, and b) sets the stage for the development of molecular protection and preventive medical nanotechnology of relevance to neurodegenerative Alzheimer's disease.

Potentiometric and Blood Plasma Simulation Studies of Nickel(II) Complexes of Poly(amino)amido Pentadentate Ligands: Computer Aided Metal-Based Drug Design.

The thermodynamic equilibria of nickel(II) with N,N'-di(aminoethylene)-2,6-pyridinedicarbonylamine (L1), Bis-(N,N-dimethylethyl)-2,6-pyridinedicarboxamide (L2), and N,N'-bis[2(2-pyridyl)-methyl]pyridine-2,6-dicarboxamide (L3) have been studied at 25°C and an ionic strength of 0.15 mol dm(-3) by glass electrode potentiometry. The protonation and formation constants added to blood plasma model predict that Cu(II) competes effectively against Ni(II), Zn(II), and Ca(II) for these ligands in vivo.

Enteric hyperoxaluria secondary to small bowel resection: use of computer simulation to characterize urinary risk factors for stone formation and assess potential treatment protocols.

BACKGROUND AND PURPOSE: We used computer modeling to investigate the influence of physicochemical stone risk factors on urinary supersaturation (SS) of calcium oxalate (CaOx) in patients with severe hyperoxaluria, relative hypocalciuria, hypocitraturia, and CaOx nephrolithiasis after extensive small bowel resection, usually performed for Crohn's disease. We also simulated different treatment strategies, including oral calcium supplements and citrate, in such patients. MATERIALS AND METHODS: A baseline urine model was derived by consolidating data acquired by ourselves with those from another patient cohort. Calcium and oxalate excretions in this model were altered to obtain an extreme case. For comparison, additional models were based on published urine data from normal subjects (N) and idiopathic CaOx stone formers (SF). The Joint Expert Speciation System was used to simulate different urine situations based on reported compositional values. RESULTS: [Ca(2+)][Ox(2-)] ionic concentration products and SS(CaOx) are substantially higher in enteric hyperoxaluric patients than in N and SF, despite their relatively lower calcium excretions. Molar Ca:Ox ratios are substantially lower in enteric hyperoxalurics than in N and SF. Oral calcium supplements can reduce SS(CaOx), but monitoring is required to avoid exceeding a safe dosing threshold. A simple calculation can alert the clinician that this threshold is being approached or even exceeded. Increasing urinary pH and citrate decreases SS(CaOx) but not to the same extent as decreasing Ox excretion. CONCLUSIONS: Calcium supplements can help reduce stone risk in patients with severe enteric hyperoxaluria, but initial efforts should be directed toward reducing urinary oxalate by reducing dietary oxalate. Citrate therapy that increases both urine pH and urinary citrate provides an additional therapeutic benefit.

Structural studies of adipokinetic hormones in water and DPC micelle solution using NMR distance restrained molecular dynamics.

Melme-CC (pGlu-Leu-Asn-Tyr-Ser-Pro-Asp-Trp amide) and Declu-CC (pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-Gly-Asn amide) are members of the insect adipokinetic hormone family with very different activities in the locust bioassay. The conformations of both peptides were determined in water and in a phospholipid (DPC) micelle solution using nuclear magnetic resonance (NMR) restrained molecular dynamics simulations. In water, Melme-CC has one dominant conformation while in DPC solution it has two preferred conformation. In water, Declu-CC has two conformations but in DPC solution it has one preferred conformation, which is similar to one of the water conformations. All the conformations have type IV ß-turn between residues 4 and 7. The binding of the two peptides to the DPC micelle is different. Melme-CC does not bind strongly to the surface and is oriented with the ß-turn facing the surface. Declu-CC interacts more strongly with the ß-turn facing away from the surface. Both termini having hydrophobic interactions with the surface. In Declu-CC the side chain of Asn(7) projects away from the chain while in Melme-CC the Asp(7) side chain is folded inside the chain. The different orientation of these side chains may account for the much higher biological activity of Declu-CC in mobilizing lipids in the locust compared to the poor biological effect of Melme-CC in this bioassay. Receptor binding of Declu-CC was tested using a model AKH receptor from Anopheles gambiae. A free energy of binding of -38.5 kJ mol(-1) was found.

Malic acid supplementation increases urinary citrate excretion and urinary pH: implications for the potential treatment of calcium oxalate stone disease.

BACKGROUND AND PURPOSE: Raising urinary pH and citrate excretion with alkali citrate therapy has been a widely used treatment in calcium nephrolithiasis. Citrate lowers ionized Ca(+2) concentrations and inhibits calcium salt precipitation. Conservative alternatives containing citrate such as fruit juices have been investigated and recommended. Any compound that induces systemic alkalosis will increase citraturia. Malate, a polycarboxylic anion like citrate, is a potential candidate for chelating Ca(+2) and for inducing systemic alkalinization. We undertook to investigate these possibilities. MATERIALS AND METHODS: Theoretical modeling of malic acid's effects on urinary Ca(+2) concentration and supersaturation (SS) of calcium salts was achieved using the speciation program JESS. Malic acid (1200 mg/day) was ingested for 7 days by eight healthy subjects. Urines (24 hours) were collected at baseline and on day 7. They were analyzed for routine lithogenic components, including pH and citrate. Chemical speciation and SS were calculated in both urines. RESULTS: Modeling showed that complexation between calcium and malate at physiological concentrations of the latter would have no effect on SS. Administration of the supplement induced statistically significant increases in pH and citraturia. The calculated concentration of Ca(+2) and concomitant SS calcium oxalate (CaOx) decreased after supplementation, but these were not statistically significant. SS for the calcium phosphate salts hydroxyapatite and tricalcium phosphate increased significantly as a consequence of the elevation in pH, but values for brushite and octacalcium phosphate did not change significantly. CONCLUSIONS: We speculate that consumption of malic acid induced systemic alkalinization leading to reduced renal tubular reabsorption and metabolism of citrate, and an increase in excretion of the latter. The decrease in SS(CaOx) was caused by enhanced complexation of Ca(+2) by citrate. We conclude that malic acid supplementation may be useful for conservative treatment of calcium renal stone disease by virtue of its capacity to induce these effects.

ß-sheet structures and dimer models of the two major tyrocidines, antimicrobial peptides from Bacillus aneurinolyticus.

The structures of two major tyrocidines, antibiotic peptides from Bacillus aneurinolyticus, in an aqueous environment were studied using nuclear magnetic resonance spectroscopy, restrained molecular dynamics (MD), circular dichroism, and mass spectrometry. TrcA and TrcC formed ß-structures in an aqueous environment. Hydrophobic and hydrophilic residues were not totally separated into nonpolar and polar faces of the peptides, indicating that tyrocidines have low amphipathicity. In all the ß-structures, residues Trp(4)/Phe(4) and Orn(9) were on the same face. The ability of the peptides to form dimers in aqueous environment was studied by replica exchange MD simulations. Both peptides readily dimerize, and predominant complex structures were characterized through cluster analysis. The peptides formed dimers by either associating sideways or stacking on top of each other. Dimers formed through sideways association were mainly stabilized by hydrogen bonding, while the other dimers were stabilized by hydrophobic interactions. The ability of tyrocidine peptides to form different types of dimers with different orientations suggests that they can form larger aggregates, as well.

Citrate occurs widely in healthy and pathological apatitic biomineral: mineralized articular cartilage, and intimal atherosclerotic plaque and apatitic kidney stones.

There is continuing debate about whether abundant citrate plays an active role in biomineralization of bone. Using solid state NMR dipolar dephasing, we examined another normally mineralized hard tissue, mineralized articular cartilage, as well as biocalcifications arising in pathological conditions, mineralized intimal atherosclerotic vascular plaque, and apatitic uroliths (urinary stones). Residual nondephasing ¹³C NMR signal at 76 ppm in the spectra of mineralized cartilage and vascular plaque indicates that a quaternary carbon atom resonates at this frequency, consistent with the presence of citrate. The presence, and as yet unproven possible mechanistic involvement, of citrate in tissue mineralization extends the compositional, structural, biogenetic, and cytological similarities between these tissues and bone itself. Out of 10 apatitic kidney stones, five contained NMR-detectable citrate. Finding citrate in a high proportion of uroliths may be significant in view of the use of citrate in urolithiasis therapy and prophylaxis. Citrate may be essential for normal biomineralization (e.g., of cartilage), play a modulatory role in vascular calcification which could be a target for therapeutic intervention, and drive the formation of apatitic rather than other calcific uroliths, including more therapeutically intractable forms of calcium phosphate.

Anopheles gambiae, Anoga-HrTH hormone, free and bound structure--a nuclear magnetic resonance experiment.

The spread of malaria by the female mosquito, Anopheles gambiae, is dependent, amongst other things, on its ability to fly. This in turn, is dependent on the adipokinetic hormone, Anoga-HrTH (pGlu-Leu-Thr-Phe-Thr-Pro-Ala-Trp-NH2). No crystal structure of this important neuropeptide is available and hence NMR restrained molecular dynamics was used to investigate its conformational space in aqueous solution and when bound to a membrane surface. The results showed that Anoga-HrTH has an almost cyclic conformation that is stabilized by a hydrogen bond between the C-terminus and Thr3. Upon docking of the agonist to its receptor, this H-bond is broken and the molecule adopts a more extended structure. Preliminary AKHR docking calculations give the free energy of binding to be -47.30 kJ/mol. There is a close correspondence between the structure of the docked ligand and literature structure-activity studies. Information about the 3D structure and binding mode of Anoga-HrTH to its receptor is vital for the design of suitable mimetics which can act as insecticides.

Open conformation of adipokinetic hormone receptor from the malaria mosquito facilitates hormone binding.

Insect flight requires rapid mobilization of energy reserves during flight, which is mediated and regulated by hormonal control via adipokinetic hormones. The structure of the G-protein receptors to which these hormones bind, are crucial in understanding many of the physiological processes in which they play a central role. To date no 3D structure of an insect G-protein coupled receptor (GPCR) is available. Here, the first models of the 3D structures of a GPCR from the malaria mosquito are presented. Homology modeling of the receptor identified from the genome of Anopheles gambiae was used to construct two models of the receptor. The 7 transmembrane helical bundles of these two models are based on the crystal structures of beta2-adrenergic receptor and rhodopsin. The flexible loop regions were modeled using high temperature simulated annealing and constrained molecular dynamic simulations. The two receptor models differ in a number of critical features, the most important of which is that the rhodopsin-based model has a 'closed' structure while the beta2-based structure is 'open'. The 'open' conformation provides easy access of the hormone to the binding pocket. Docking calculations with the insect adipokinetic hormones, AKH-1 (pGlu-Leu-Thr-Phe-Thr-Pro-Ala-Trp-NH(2)) from the malaria mosquito and Del-CC (pGlu-Lys-Asn-Phe-Ser-Pro-Asn-Trp-Gly-Asn-NH(2)) from the blister beetle showed that while the binding motif of the two is similar, AKH-1 has more than 30 times higher affinity than Del-CC, which strongly suggests that the binding is specific, and that the correct binding site was identified. Using these models it is possible to design antagonists, which block the binding site and are thus species-specific insecticides.