IRAK3 modulates downstream innate immune signalling through its guanylate cyclase activity.

Interleukin-1 receptor associated kinase 3 (IRAK3) is a cytoplasmic homeostatic mediator of inflammatory responses and is potentially useful as a prognostic marker in inflammation. IRAK3 inhibits signalling cascades downstream of myddosome complexes associated with toll like receptors. IRAK3 contains a death domain that interacts with other IRAK family members, a pseudokinase domain and a C-terminus domain involved with tumour necrosis factor receptor associated factor 6 (TRAF6). Previous bioinformatic studies revealed that IRAK3 contained a guanylate cyclase centre in its pseudokinase domain but its role in IRAK3 action is unresolved. We demonstrate that wildtype IRAK3 is capable of producing cGMP. Furthermore, we show that a specific point mutation in the guanylate cyclase centre reduced cGMP production. Cells containing toll like receptor 4 and a nuclear factor kappa-light-chain-enhancer of activated B cells (NFĸB) reporter system were transfected with IRAK3 or mutant IRAK3 proteins. Cell-permeable cGMP treatment of untransfected control cells suppresses downstream signalling through modulation of the NFĸB in the presence of lipopolysaccharides. Cells transfected with wildtype IRAK3 also suppress lipopolysaccharide induced NFĸB activity in the absence of exogenous cGMP. Lipopolysaccharide induced NFĸB activity was not suppressed in cells transfected with the IRAK3 mutant with reduced cGMP-generating capacity. Whereas in the presence of exogenously applied cell-permeable cGMP the IRAK3 mutant was able to retain its function by suppressing lipopolysaccharide induced NFĸB activity. Furthermore, increasing the amount of membrane permeable cGMP did not affect IRAK3's ability to reduce NFĸB activity. These results suggest that cGMP generated by IRAK3 may be involved in regulatory function of the protein where the presence of cGMP may selectively affect downstream signalling pathway(s) by modulating binding and/or activity of nearby proteins that interact in the inflammatory signalling cascade.

Platensimycin: a promising antimicrobial targeting fatty acid synthesis.

Platensimycin was recently discovered by Merck Research Laboratories and has created considerable interest given its potent antibacterial activity and mode of action. The use of RNA gene-silencing techniques and screening libraries of natural products allowed Merck to find this antibiotic which may have otherwise been missed using conventional methods. Interestingly, platensimycin has shown good activity against a panel of Gram positive organisms which included various resistant strains. Platensimycin works by inhibiting beta-ketoacyl synthases I/II (FabF/B) which are key enzymes in the production of fatty acids required for bacterial cell membranes. So far, a number of groups have explored synthetic strategies for platensimycin and this work has subsequently lead to the synthesis of active analogues. Given its mode of action, it is intriguing as to why Merck themselves patented only a single compound and have not apparently sought to generate further libraries. This review will discuss the origins of platensimycin, its mechanism of action, synthetic schemes and where the future may take us following this fascinating discovery.

Prediction of drug solubility from molecular structure using a drug-like training set.

Using a training set of 191 drug-like compounds extracted from the AQUASOL database a quantitative structure-property relationship (QSPR) study was conducted employing a set of simple structural and physicochemical properties to predict aqueous solubility. The resultant regression model comprised five parameters (ClogP, molecular weight, indicator variable for aliphatic amine groups, number of rotatable bonds and number of aromatic rings) and demonstrated acceptable statistics (r2 = 0.87, s = 0.51, F = 243.6, n = 191). The model was applied to two test sets consisting of a drug-like set of compounds (r2 = 0.80, s = 0.68, n = 174) and a set of agrochemicals (r2 = 0.88, s = 0.65, n = 200). Using the established general solubility equation (GSE) on the training and drug-like test set gave poorer results than the current study. The agrochemical test set was predicted with equal accuracy using the GSE and the QSPR equation. The results of this study suggest that increasing molecular size, rigidity and lipophilicity decrease solubility whereas increasing conformational flexibility and the presence of a non-conjugated amine group increase the solubility of drug-like compounds. Indeed, the proposed structural parameters make physical sense and provide simple guidelines for modifying solubility during lead optimisation.

The acid/base profile of agrochemicals.

Drug-likeness has long been studied in the pursuit of finding new medicines. Similarly, in the agrochemical field there is value in exploring the properties of the chemicals involved. Patterns that emerge can potentially influence future discovery campaigns to improve the probability of commercial success. In this study we investigate the acid/base properties and physicochemical characteristics of three classes of compounds: fungicides, herbicides and insecticides. In comparison with FDA-approved drugs, it was noted that the pesticides were generally smaller, possessed a neutral charge state and were more lipophilic. The results are discussed in the light of their intended targets.

Absence of N-methyl-D-aspartate receptors on ovine cerebral microvessels.

Radioreceptor methods were used to quantitate the N-methyl-D-aspartate (NMDA) receptor-complex of ovine cerebral microvessels and cerebral gray matter. Specific binding of D[3H]2-amino-5-phosphono-pentanoate and [3H]1-[1-(2-thienyl)cyclohexyl]piperidine, ligands for the NMDA primary acceptor site and ionophore, respectively, was found in cerebral gray matter but was not detectable in membranes prepared from brain microvessels enriched in capillaries. Sigma receptors, another locus of action for phencyclidine congeners, were also not present on microvessels but were found in cortical homogenates. On the other hand, cerebral microvessels and gray matter contained significant numbers of beta-adrenoceptors. Our results indicate the NMDA receptors and NMDA antagonists are unlikely to regulate the function of the cerebral microvasculature.

Up-regulation of rat cortical sigma receptors after subchronic administration of rimcazole and 1,3-di(2-tolyl)guanidine.

The effects of subchronic administration of rimcazole and 1,3-di(2-tolyl)guanidine (DTG) on the central R(+)[3H]-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine (R(+)[3H]-3-PPP) binding site were investigated in the rat. Subchronic treatment with rimcazole was characterized by a 30% increase in the density and a two-fold decrease in the affinity of R(+)[3H]-3-PPP binding sites relative to saline-treated animals. DTG, a more potent sigma ligand, produced a similar alteration to the affinity but a 130% increase in the density of R(+)[3H]-3-PPP binding sites. These data thus provide evidence for the functional involvement of sigma receptors in the central nervous system.

Design, synthesis, and testing of insulin hexamer-stabilizing agents.

The addition of zinc to insulin solution leads to a long-acting insulin preparation because the zinc stabilizes the less soluble hexameric form of the hormone. It is clear from the crystal structure of dizinc insulin that there is a space at the center of the hexamer, between the two zinc atoms, that could accommodate a small organic molecule. It should thus be possible to design a structure that could further stabilize the insulin hexamer by binding at this site. Computer graphic techniques have been used to design several molecules capable of forming multiple bonds to the six histidine residues surrounding the site. Synthesis and testing of one of these compounds, benzene-1,4-disulfonic acid, show a significant increase in weight-average molecular weight of insulin in solution, and control experiments with related structures suggest that this effect is due to the proposed binding mechanism.

Analysis of linear and nonlinear QSAR data using neural networks.

The use of feed forward back propagation neural networks to perform the equivalent of multiple linear regression has been examined using artificial structured data sets and real literature data. Their predictive ability has been assessed using leave-one-out cross-validation and training/test set protocols. While networks have been shown to fit data sets well, they appear to suffer from a number of disadvantages. In particular, they have performed poorly in prediction for the QSAR data examined here, they are susceptible to chance effects, and the relationships developed by the networks are difficult to interpret. This investigation reports results for one particular form of artificial neural network; other architectures and applications, however, may be more suitable.

Subchronic administration of MK-801 in the rat decreases cortical binding of [3H]D-AP5, suggesting down-regulation of the cortical N-methyl-D-aspartate receptors.

The effects of the subchronic administration of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine (MK-801) (0.5 mg/kg twice daily, 7 days) on N-methyl-D-aspartate, phencyclidine and sigma binding sites, behaviour and catecholamine turnover were investigated in the rat. Overt behaviours induced by MK-801 on day 7 were significantly altered relative to day 1 with subchronically treated rats not showing head weaving, goss ataxia or loss of hindlimb control: locomotion and sniffing were largely unaffected. The mean intensities of behaviour were 1.8 and 5.4 for days 7 and 1, respectively. Behavioural tolerance was accompanied by a significant reduction in the density of cortical N-methyl-D-aspartate receptors as measured by [3H]D-2-amino-5-phosphonopentanoic acid binding, while affinity was unchanged: the density of binding sites was 3.52 and 1.88 pmol/mg protein for saline- and MK-801-treated rats, respectively. The N-methyl-D-aspartate ion channel as measured by the binding of [3H]N-(1-[2-thienyl]cyclohexyl)piperidine was not affected by the schedule of MK-801. Additionally, changes were not observed to N-methyl-D-aspartate- or glycine-stimulated [3H]N-(1-[2-thienyl]cyclohexyl)piperidine binding or to sigma binding. Catecholamine turnover was unaltered in the nucleus accumbens septi after the schedule of MK-801. Our results demonstrate that the subchronic administration of MK-801 produces behavioural tolerance and down-regulation of N-methyl-D-aspartate binding sites and suggest differential regulation of the domains of the N-methyl-D-aspartate receptor-ionophore complex.

A comparison between the in vivo and in vitro activity of five potent and competitive NMDA antagonists.

1. Phosphonate analogues of glutamate have been tested and compared as N-methyl-D-aspartate (NMDA) antagonists in electrophysiological and binding experiments. The compounds tested were three established NMDA antagonists: D-2-amino-5-phosphonopentanoate (D-AP5), DL-2-amino-7-phosphonoheptanoate (DL-AP7), 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), and two novel putative NMDA antagonists: 3-(2-carboxypiperidin-4-yl)propyl-1-phosphonate (CPPP) and 3-(2-carboxy-piperidin-4-yl)methyl-1-phosphonate (CPMP). 2. When administered electrophoretically to rat spinal neurones in vivo, these compounds were found to be selective NMDA antagonists with little effect on excitations evoked by quisqualate and kainate. CPMP and CPPP were approximately equipotent with CPP and about 5 times more potent than D-AP5. 3. Following systemic administration, 2-5 mg kg-1 i.v. of CPP, CPMP and CPPP reduced NMDA-evoked excitations by 70-100% whereas 50-100 mg kg-1 of D-AP5 and DL-AP7 produced a similar effect. The onset of the effects required 20-30 min and lasted more than six hours. 4. On bath application to cortical wedges, the IC50 values (microM) for antagonism of 40 microM NMDA were: CPP, 0.64 +/- 0.06 (mean +/- s.e.mean; n greater than 4); CPMP, 1.65 +/- 0.13; CPPP 0.89 +/- 0.09; D-AP5, 3.7 +/- 0.32; DL-AP7, 11.1 +/- 2.1; and DL-AP4 and DL-AP6 were inactive at 100 microM. 5. In binding studies with [3H]-CPP, the Ki values (nM) were: CPP, 446 + 150 (mean + s.e.mean; n > 3); CPMP, 183 + 74 and CPPP, 179 +/- 13 whereas against NMDA (lO microM)-stimulated [3H]- TCP (thienylcyclohexylpiperidine) binding the ICjo values (microM) for CPMP and CPPP respectively were 5.6 + 2.7 and 4.5 + 2.2. 6. Systemic administration of CPPP and CPMP, at doses sufficient to antagonize NMDA, also reduced cardiovascular responses to 5-hydroxytryptamine (Bezold-Jarisch reflex). This illustrates a role for NMDA receptors in central cardiovascular control. 7. The results indicate the systemic doses of piperidine and piperazine analogues of D-AP5 which may be used for assessing the role ofNMDA receptors in central synaptic function.

Quantitative conformational analyses predict distinct receptor sites for PCP-like and sigma drugs.

Computer-assisted molecular modelling techniques have been employed to develop receptor models for the phencyclidine (PCP) and sigma binding sites. The models differ in the position of the nitrogen atom, direction of the nitrogen-lone pair vector and in the location and nature of secondary binding groups. This study predicts the existence of distinct receptors for sigma and PCP ligands, and our receptor models can be used to design and predict the activity of drugs with PCP and/or sigma activity.

Ionic regulation of the binding of DL-2-amino-7-phosphono-[4,5-3H]heptanoic acid to synaptosome-enriched homogenates of rat cerebral cortex.

The ionic requirements of the site labelled by DL-2-amino-7-phosphono-[4,5-3H]heptanoic acid ([3H]DL-2AP7) in synaptosome-enriched homogenates of rat cerebral cortex were examined using radioligand binding methodology. Binding of [3H]DL-2AP7 was increased by calcium and chloride ions by an apparently non-competitive mechanism. The actions of ions on specific binding displayed similarities to the effects of ions on DL-[3H]2-amino-4-phosphonobutyric acid and Cl-/Ca2+-dependent L-[3H]glutamate ([3H]Glu) binding sites but were more consistent with the Glu-C binding site.

Theoretical hydrogen bonding parameters for drug design.

Hydrogen bonding interactions play a major role in many chemical and biological processes. This article describes the development of a method for the quantitative estimation of the hydrogen-bonding donor strengths of OH/NH moieties and of the hydrogen bonding acceptor strengths of O/N atoms in different chemical structures. The method is based on the correlation of experimentally observed hydrogen-bonding strengths with quantum-mechanical derived properties, calculated on the acceptor atom (for hydrogen-bond acceptors) and on the heavy atom attached to the donor hydrogen (for hydrogen-bond donors). The properties giving the best correlation with the experimental hydrogen bonding scales were electrophilic superdelocalizability and self-atom polarizability. The best equations found have been implemented in a Web-based tool for hydrogen-bond strength prediction.

Multivariate quantitative structure-toxicity relationships in a series of dopamine mimetics.

The techniques of principal components analysis and non-linear mapping are routinely used by computer chemists at SmithKline Beecham Pharmaceuticals in the process of drug development by relating the structure of a compound to its chemical activity. To our knowledge these techniques had not previously been applied to the association between the structure of a compound and its toxicological properties. Using a series of 12 structurally related compounds (11 were active dopamine mimetics and one was inactive), of which five were known to be teratogenic and seven were non-teratogenic, it was possible to demonstrate that molecular modelling techniques could be applied to differentiate toxicological data. The structure/property relationships of these compounds were investigated using calculated physicochemical properties, molecular modelling and multivariate statistical techniques. A data set of 56 molecular descriptors was used to represent this series of compounds. Analysis of the data set using principal components analysis and non-linear mapping suggested that teratogenicity was associated with four molecular properties. Moreover, the electronic nature of the 4-phenyl group appeared to be an important determinant of the teratogenesis.

A three dimensional receptor model of the dopamine D2 receptor from computer graphic analyses of D2 agonists.

Four potent D2 agonists were employed to define a primary pharmacophore for the D2 receptor. Hypothetical receptor points, representing interaction points on a receptor were built on to each molecule. These points and the nitrogen atom were averaged to give the coordinates (A) of the primary pharmacophore: R1 (0.00, 3.50, 0.00), R2 (0.00, -3.50, 0.00), R3 (5.79, 2.06, 0.00), and nitrogen (5.13, -0.63, 0.37). Eight structural classes of D2 agonists were then superimposed on to the primary pharmacophore to aid in the location of secondary binding sites. The secondary sites include two lipophilic clefts, an area of steric bulk, a region to hydrogen bond 'meta' hydroxy groups and a 'critical region' accepting methoxy and halogen substituents but not hydroxy substituents. The model has the potential to design and predict activity of novel D2 agonist compounds.

Use of Kv1.3 blockers for inflammatory skin conditions.

Recent results using animal models of inflammatory skin conditions have shown that blockers of the voltage-gated potassium channel, Kv1.3 hold great promise for clinical utility. Kv1.3 blockers act as immunosuppressants by modulating the various subsets of inflammatory T and B cells involved in autoimmune disorders. While peptidic inhibitors based on naturally occurring venoms demonstrate potent and selective Kv1.3 blockade, these require parenteral administration and may face potential immunogenicity problems. Small molecule blockers show considerable diversity, however selectivity over other Kv1-family channels has been difficult to achieve. More recent advances have added to the evidence that Kv1.3 channels are a suitable therapeutic target and that the development of novel and selective agents will herald new drugs for inflammatory skin disorders.

Synthesis and preliminary screening of novel tryptamines as 5-HT4 receptor ligands.

For the development of novel 5-HT(4) receptor ligands we have designed and synthesized two series of 5-methoxytryptamine derivatives varying the substitution on the primary amine. Their biological activities were evaluated in a receptor binding assay where a subset of compounds showed comparable potency to the agonists serotonin and 5-methoxytryptamine. Structure-activity analyses have highlighted promising avenues for further synthetic work and binding modes were proposed by docking these compounds into a homology model of the 5-HT(4) receptor.

The acid-base profile of a contemporary set of drugs: implications for drug discovery.

Acid-base ionization constant (pK(a)) values have considerable influence on the physicochemical and pharmacokinetic properties of therapeutic substances. A set of 907 drugs was examined to determine the proportion of drugs that contain an ionizable group and the distribution of their pK(a) values. Using this contemporary set of compounds it was found that 64% of these compounds contained an ionizable group. Within this group of ionizable compounds, 34% contained a single basic group while only 20% contained a single acidic functional group. The single acid and single base containing substances were investigated further to examine the distributions of their pK(a) values. These data are discussed and analyzed with a focus on the entire set as well as central nervous system, non-central nervous system and oral drugs. The findings from this research will prompt pharmaceutical companies to assess the constitution of their screening libraries, such that focus is placed on the proportion of ionizable substances, the ratio of acids to bases and the distribution of pK(a) values.

The use of tripeptides for lead discovery of 5-HT4 receptor ligands.

A series of 30 tripeptides were synthesized and tested as novel 5-HT4 receptor ligands. Receptor binding assays showed that a subset of compounds had reasonable potency relative to the agonists serotonin and 5-methoxytryptamine. Structure-activity analyses and molecular docking have highlighted avenues for further synthetic work.