The discovery of hidden guanylate cyclases (GCs) in the Homo sapiens proteome.

Recent discoveries have established functional guanylate cyclase (GC) catalytic centers with low activity within kinase domains in plants. These crypto GCs generate guanosine 3',5'-cyclic monophosphate (cGMP) essential for both intramolecular and downstream signaling. Here, we have set out to search for such crypto GCs moonlighting in kinases in the H. sapiens proteome and identified 18 candidates, including the neurotropic receptor tyrosine kinase 1 (NTRK1). NTRK1 shows a domain architecture much like plant receptor kinases such as the phytosulfokine receptor, where a functional GC essential for downstream signaling is embedded within a kinase domain. In vitro characterization of the NTRK1 shows that the embedded NTRK1 GC is functional with a marked preference for Mn2+ over Mg2+. This therefore points to hitherto unsuspected roles of cGMP in intramolecular and downstream signaling of NTRK1 and the role of cGMP in NTRK1-dependent growth and neoplasia.

Mutations in the Vicinity of the IRAK3 Guanylate Cyclase Center Impact Its Subcellular Localization and Ability to Modulate Inflammatory Signaling in Immortalized Cell Lines.

Interleukin-1 receptor-associated kinase 3 (IRAK3) modulates the magnitude of cellular responses to ligands perceived by interleukin-1 receptors (IL-1Rs) and Toll-like receptors (TLRs), leading to decreases in pro-inflammatory cytokines and suppressed inflammation. The molecular mechanism of IRAK3's action remains unknown. IRAK3 functions as a guanylate cyclase, and its cGMP product suppresses lipopolysaccharide (LPS)-induced nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) activity. To understand the implications of this phenomenon, we expanded the structure-function analyses of IRAK3 through site-directed mutagenesis of amino acids known or predicted to impact different activities of IRAK3. We verified the capacity of the mutated IRAK3 variants to generate cGMP in vitro and revealed residues in and in the vicinity of its GC catalytic center that impact the LPS-induced NFκB activity in immortalized cell lines in the absence or presence of an exogenous membrane-permeable cGMP analog. Mutant IRAK3 variants with reduced cGMP generating capacity and differential regulation of NFκB activity influence subcellular localization of IRAK3 in HEK293T cells and fail to rescue IRAK3 function in IRAK3 knock-out THP-1 monocytes stimulated with LPS unless the cGMP analog is present. Together, our results shed new light on the mechanism by which IRAK3 and its enzymatic product control the downstream signaling, affecting inflammatory responses in immortalized cell lines.

Visualising functional 5-HT3 receptors containing A and C subunits at or near the cell surface.

Five different subunits of the human serotonin 3 (5-hydroxytrptamine 3; 5-HT3) receptor exist and these are present in both central and peripheral systems. Different subunits alter the efficacy of 5-HT3 receptor antagonists used to treat diarrhoea predominant-irritable bowel syndrome, chemotherapy induced nausea and vomiting and depression. Cell surface arrangement of 5-HT3 receptor complexes and the contribution of C, D and E subunits to receptor function is poorly understood. Here, we examine interactions of A and C subunits using 5-HT3 receptor subunits containing fluorescent protein inserts between the 3rd and 4th transmembrane spanning region. HEK293T cells that do not normally express 5-HT3 receptor subunits, were transiently transfected with A or C or both subunits. Patch clamp experiments show that cells transfected with either fluorescent protein tagged A or A and C subunits generate whole cell currents in response to 5-HT. These findings correlate with the apparent distribution of fluorescent protein tagged A and C subunits at or near cell surfaces detected using TIRF microscopy. In co-transfected cells, the A and C subunits are associated forming AC heteromer complexes at or near the cell surface and a proportion can also form A or C homomers. In conclusion, it is likely that both A homomers and AC heteromers contribute to whole cell currents in response to 5-HT with minimal contribution from C homomers.

The Acid/Base Profile of a Large Food Chemical Database.

Molecular acid/base properties have a significant influence on membrane permeation, metabolism, absorption, and affinity for biological targets. In particular, ionizable groups are critical in the strength of target-molecule interactions, pharmacokinetics, and toxicity. In this study, we estimated the acid/base properties of the food chemicals from FooDB, a public compound collection with more than 22,000 compounds. It was found that the food chemicals have 40.9 % of neutral compounds, which is twice as many as that found in approved drugs. The most common functional groups among the acid groups in the food chemicals were phenols (16.1 %), phosphates (17.3 %), and carboxylates (17.3 %) while the single-base-containing compounds were of less interest as they accounted for just 5.5 %. To the best of our knowledge, this is the first systematic acid/base profiling of food chemicals and it is part of a continued effort to profile food chemicals for their broad interest in several areas such as nutrition and the food industry in general.

The influence and manipulation of acid/base properties in drug discovery.

There is a growing awareness of the importance of acid/base properties in medicinal chemistry research. In many drug classes, ionisable groups are present that make critical interactions with the receptor and are essential for potency. Yet the presence of these groups may cause problems with oral bioavailability, pharmacokinetics, or toxicity. Manipulating pKa values during drug development or applying pro-drug techniques are strategies that can overcome potential deficits in a variety of these areas. Knowledge of drug ionisation states coupled with a consideration of pH-specific cellular environments can be used advantageously to target chemoresistance. As modern drug research ventures into drug candidates that exceed the rule of 5, such exploration requires an understanding of drug acid/base properties and how these factors affect ADMET characteristics.

Inhibition of DYRK1A disrupts neural lineage specificationin human pluripotent stem cells.

Genetic analysis has revealed that the dual specificity protein kinase DYRK1A has multiple roles in the development of the central nervous system. Increased DYRK1A gene dosage, such as occurs in Down syndrome, is known to affect neural progenitor cell differentiation, while haploinsufficiency of DYRK1A is associated with severe microcephaly. Using a set of known and newly synthesized DYRK1A inhibitors, along with CRISPR-mediated gene activation and shRNA knockdown of DYRK1A, we show here that chemical inhibition or genetic knockdown of DYRK1A interferes with neural specification of human pluripotent stem cells, a process equating to the earliest stage of human brain development. Specifically, DYRK1A inhibition insulates the self-renewing subpopulation of human pluripotent stem cells from powerful signals that drive neural induction. Our results suggest a novel mechanism for the disruptive effects of the absence or haploinsufficiency of DYRK1A on early mammalian development, and reveal a requirement for DYRK1A in the acquisition of competence for differentiation in human pluripotent stem cells.

Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1.

Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1) that also contains a functional cytosolic guanylate cyclase with its cyclase catalytic center embedded within the kinase domain. To functionally characterize this novel type of overlapping dual catalytic function, we investigated the phosphorylation of PSKR1 in vitro Tandem mass spectrometry of the cytoplasmic domain of PSKR1 (PSKR1cd) revealed at least 11 phosphorylation sites (8 serines, 2 threonines and 1 tyrosine) within the PSKR1cd. Phosphomimetic mutations of three serine residues (Ser686, Ser696 and Ser698) in tandem at the juxta-membrane position resulted in enhanced kinase activity in the on-mutant that was suppressed in the off-mutant, but both mutations reduced guanylate cyclase activity. Both the on and off phosphomimetic mutations of the phosphotyrosine (Tyr888) residue in the activation loop suppressed kinase activity, while neither mutation affected guanylate cyclase activity. Size exclusion and analytical ultracentrifugation analysis of the PSKR1cd suggest that it is reversibly dimeric in solution, which was further confirmed by biflourescence complementation. Taken together, these data suggest that in this novel type of receptor domain architecture, specific phosphorylation and dimerization are possibly essential mechanisms for ligand-mediated catalysis and signaling.

Developing a Framework for Objective Structured Clinical Examinations Using the Nominal Group Technique.

Objective. To use the nominal group technique to develop a framework to improve existing and develop new objective structured clinical examinations (OSCEs) within a four-year bachelor of pharmacy course. Design. Using the nominal group technique, a unique method of group interview that combines qualitative and quantitative data collection, focus groups were conducted with faculty members, practicing pharmacists, and undergraduate pharmacy students. Five draft OSCEs frameworks were suggested and participants were asked to generate new framework ideas. Assessment. Two focus groups (n=9 and n=7) generated nine extra frameworks. Two of these frameworks, one from each focus group, ranked highest (mean scores of 4.4 and 4.1 on a 5-point scale) and were similar in nature. The project team used these two frameworks to produce the final framework, which includes an OSCE in every year of the course, earlier implementation of teaching OSCEs, and the use of independent simulated patients who are not examiners. Conclusions. The new OSCE framework provides a consistent structure from course entry to exit and ensures graduates meet internship requirements.

Identification of potent phosphodiesterase inhibitors that demonstrate cyclic nucleotide-dependent functions in apicomplexan parasites.

Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, the causative agents of severe malaria and toxoplasmosis, respectively, undergo several critical developmental transitions during their lifecycle. Most important for human pathogenesis is the asexual cycle, in which parasites undergo rounds of host cell invasion, replication, and egress (exit), destroying host cell tissue in the process. Previous work has identified important roles for Protein Kinase G (PKG) and Protein Kinase A (PKA) in parasite egress and invasion, yet little is understood about the regulation of cyclic nucleotides, cGMP and cAMP, that activate these enzymes. To address this, we have focused upon the development of inhibitors of 3',5'-cyclic nucleotide phosphodiesterases (PDEs) to block the breakdown of cyclic nucleotides. This was done by repurposing human PDE inhibitors noting various similarities of the human and apicomplexan PDE binding sites. The most potent inhibitors blocked the in vitro proliferation of P. falciparum and T. gondii more potently than the benchmark compound zaprinast. 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO) was found to be a potent inhibitor of recombinant P. falciparum PfPDEα and activated PKG-dependent egress of T. gondii and P. falciparum, likely by promoting the exocytosis of micronemes, an activity that was reversed by a specific Protein Kinase G inhibitor. BIPPO also promotes cAMP-dependent phosphorylation of a P. falciparum ligand critical for host cell invasion, suggesting that the compound inhibits single or multiple PDE isoforms that regulate both cGMP and cAMP levels. BIPPO is therefore a useful tool for the dissection of signal transduction pathways in apicomplexan parasites.

Comparison of moonlighting guanylate cyclases: roles in signal direction?

Over 30 receptor-like kinases contain a guanylate cyclase (GC) catalytic centre embedded within the C-terminal region of their kinase domain in the model plant Arabidopsis. A number of the kinase GCs contain both functional kinase and GC activity in vitro and the natural ligands of these receptors stimulate increases in cGMP within isolated protoplasts. The GC activity could be described as a minor or moonlighting activity. We have also identified mammalian proteins that contain the novel GC centre embedded within kinase domains. One example is the interleukin 1 receptor-associated kinase 3 (IRAK3). We compare the GC functionality of the mammalian protein IRAK3 with the cytoplasmic domain of the plant prototype molecule, the phytosulfokine receptor 1 (PSKR1). We have developed homology models of these molecules and have undertaken in vitro experiments to compare their functionality and structural features. Recombinant IRAK3 produces cGMP at levels comparable to those produced by PSKR1, suggesting that IRAK3 contains GC activity. Our findings raise the possibility that kinase-GCs may switch between downstream kinase-mediated or cGMP-mediated signalling cascades to elicit desired outputs to particular stimuli. The challenge now lies in understanding the interaction between the GC and kinase domains and how these molecules utilize their dual functionality within cells.

Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1.

BACKGROUND: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. FINDINGS: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. CONCLUSIONS: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

Homology modeling of human muscarinic acetylcholine receptors.

We have developed homology models of the acetylcholine muscarinic receptors M1R-M5R, based on the ß2-adrenergic receptor crystal as the template. This is the first report of homology modeling of all five subtypes of acetylcholine muscarinic receptors with binding sites optimized for ligand binding. The models were evaluated for their ability to discriminate between muscarinic antagonists and decoy compounds using virtual screening using enrichment factors, area under the ROC curve (AUC), and an early enrichment measure, LogAUC. The models produce rational binding modes of docked ligands as well as good enrichment capacity when tested against property-matched decoy libraries, which demonstrates their unbiased predictive ability. To test the relative effects of homology model template selection and the binding site optimization procedure, we generated and evaluated a naïve M2R model, using the M3R crystal structure as a template. Our results confirm previous findings that binding site optimization using ligand(s) active at a particular receptor, i.e. including functional knowledge into the model building process, has a more pronounced effect on model quality than target-template sequence similarity. The optimized M1R-M5R homology models are made available as part of the Supporting Information to allow researchers to use these structures, compare them to their own results, and thus advance the development of better modeling approaches.

A chemogenomic analysis of ionization constants--implications for drug discovery.

Chemogenomics methods seek to characterize the interaction between drugs and biological systems and are an important guide for the selection of screening compounds. The acid/base character of drugs has a profound influence on their affinity for the receptor, on their absorption, distribution, metabolism, excretion and toxicity (ADMET) profile and the way the drug can be formulated. In particular, the charge state of a molecule greatly influences its lipophilicity and biopharmaceutical characteristics. This study investigates the acid/base profile of human small-molecule drugs, chemogenomics datasets and screening compounds including a natural products set. We estimate the acid-ionization constant (pK(a)) values of these compounds and determine the identity of the ionizable functional groups in each set. We find substantial differences in acid/base profiles of the chemogenomic classes. In many cases, these differences can be linked to the nature of the target binding site and the corresponding functional groups needed for recognition of the ligand. Clear differences are also observed between the acid/base characteristics of drugs and screening compounds. For example, the proportion of drugs containing a carboxylic acid was 20 %, in stark contrast to a value of 2.4 % for the screening set sample. The proportion of aliphatic amines was 27 % for drugs and only 3.4 % for screening compounds. This suggests that there is a mismatch between commercially available screening compounds and the compounds that are likely to interact with a given chemogenomic target family. Our analysis provides a guide for the selection of screening compounds to better target specific chemogenomic families with regard to the overall balance of acids, bases and pK(a) distributions.

The significance of acid/base properties in drug discovery.

While drug discovery scientists take heed of various guidelines concerning drug-like character, the influence of acid/base properties often remains under-scrutinised. Ionisation constants (pK(a) values) are fundamental to the variability of the biopharmaceutical characteristics of drugs and to underlying parameters such as logD and solubility. pK(a) values affect physicochemical properties such as aqueous solubility, which in turn influences drug formulation approaches. More importantly, absorption, distribution, metabolism, excretion and toxicity (ADMET) are profoundly affected by the charge state of compounds under varying pH conditions. Consideration of pK(a) values in conjunction with other molecular properties is of great significance and has the potential to be used to further improve the efficiency of drug discovery. Given the recent low annual output of new drugs from pharmaceutical companies, this review will provide a timely reminder of an important molecular property that influences clinical success.

The Acid/Base Profile of the Human Metabolome and Natural Products.

Human small molecule metabolites (the human metabolome) are a set of compounds that interact with at least one macromolecule in the biosphere. This study investigates the acid/base profile of the human metabolome, natural products and drugs, together with an analysis of their physicochemical properties. Ionisation constants (pKa values) are estimated for each compound and the identity of the ionisable functional groups in each set is determined. The acid/base and physicochemical property profile of the lipid component of the metabolome differed considerably to the other datasets. In contrast, the acid/base properties of non-lipid metabolites were found to be similar to both drugs and natural products. While the non-lipid metabolites have lower average ClogP values and more hydrogen bond donors than the other datasets, the distribution of physicochemical property values overlapped considerably with the drug dataset. Considering also that the non-lipid metabolites are of biochemical interest, their characteristics have great potential to influence the selection of screening compounds for drug discovery.

Structure-activity relationship exploration of Kv1.3 blockers based on diphenoxylate.

Diphenoxylate, a well-known opioid agonist and anti-diarrhoeal agent, was recently found to block Kv1.3 potassium channels, which have been proposed as potential therapeutic targets for a range of autoimmune diseases. The molecular basis for this Kv1.3 blockade was assessed by the selective removal of functional groups from the structure of diphenoxylate as well as a number of other structural variations. Removal of the nitrile functional group and replacement of the C-4 piperidinyl substituents resulted in several compounds with submicromolar IC(50) values.

Assessment of the pharmacological properties of 5-methoxyindole derivatives at 5-HT4 receptors.

OBJECTIVES: The aim was to examine the biological activity of 5-methoxytryptamine derivatives at the 5-hydroxytryptamine (5-HT)(4) receptor to explore the effect of substitution on the aliphatic amine of the 5-methoxyamine scaffold. METHODS: Three compounds were tested for affinity at the 5-HT(4) receptor by radioligand binding and functional activity using guinea-pig ileum and human colon circular muscle preparations and also in the mouse whole gut transit test. KEY FINDINGS: The three compounds all had agonist properties at the 5-HT(4) receptor but their efficacy differed in the different functional tests. Compound 3 had the highest affinity for the 5-HT(4) receptor and was a full agonist at relaxing human colon circular muscle with efficacy closest to 5-HT. Compounds 1 and 2 were partial agonists in this assay with lower efficacies; compound 2 was a full agonist in the guinea-pig ileum assay whereas compound 3 was a partial agonist. Compounds 1 and 2 also showed activity in the mouse gut transit assay while compound 3 had no activity. CONCLUSIONS: Of the compounds tested, compound 3 was the most promising 5-HT(4) receptor agonist and the results highlight the value of using human tissue in functional tests when assessing compounds for potential activity.

Thiophene inhibitors of PDE4: crystal structures show a second binding mode at the catalytic domain of PDE4D2.

PDE4 inhibitors have been identified as therapeutic targets for a variety of conditions, particularly inflammatory diseases. We have serendipitously identified a novel class of phosphodiesterase 4 (PDE4) inhibitor during a study to discover antagonists of the parathyroid hormone receptor. X-ray crystallographic studies of PDE4D2 complexed to four potent inhibitors reveal the atomic details of how they inhibit the enzyme and a notable contrast to another recently reported thiophene-based inhibitor.

Active site similarity between human and Plasmodium falciparum phosphodiesterases: considerations for antimalarial drug design.

The similarity between Plasmodium falciparum phosphodiesterase enzymes (PfPDEs) and their human counterparts have been examined and human PDE9A was found to be a suitable template for the construction of homology models for each of the four PfPDE isoforms. In contrast, the architecture of the active sites of each model was most similar to human PDE1. Molecular docking was able to model cyclic guanosine monophosphate (cGMP) substrate binding in each case but a docking mode supporting cyclic adenosine monophosphate (cAMP) binding could not be found. Anticipating the potential of PfPDE inhibitors as anti-malarial drugs, a range of reported PDE inhibitors including zaprinast and sildenafil were docked into the model of PfPDEα. The results were consistent with their reported biological activities, and the potential of PDE1/9 inhibitor analogues was also supported by docking.