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Protein–protein interactions (PPIs) are essential for the function of many proteins. Aberrant PPIs have the potential to lead to disease, making PPIs promising targets for drug discovery. There are over 64,000 PPIs in the human interactome reference database; however, to date, very few PPI modulators have been approved for clinical use. Further development of PPI-specific therapeutics is highly dependent on the availability of structural data and the existence of reliable computational tools to explore the interface between two interacting proteins. The fragment molecular orbital (FMO) quantum mechanics method offers comprehensive and computationally inexpensive means of identifying the strength (in kcal/mol) and the chemical nature (electrostatic or hydrophobic) of the molecular interactions taking place at the protein–protein interface. We have integrated FMO and PPI exploration (FMO-PPI) to identify the residues that are critical for protein–protein binding (hotspots). To validate this approach, we have applied FMO-PPI to a dataset of protein–protein complexes representing several different protein subfamilies and obtained FMO-PPI results that are in agreement with published mutagenesis data. We observed that critical PPIs can be divided into three major categories: interactions between residues of two proteins (intermolecular), interactions between residues within the same protein (intramolecular), and interactions between residues of two proteins that are mediated by water molecules (water bridges). We extended our findings by demonstrating how this information obtained by FMO-PPI can be utilized to support the structure-based drug design of PPI modulators (SBDD-PPI).

G-protein coupled receptors (GPCRs) are the largest superfamily of membrane proteins, regulating almost every aspect of cellular activity and serving as key targets for drug discovery. We have identified an accurate and reliable computational method to characterize the strength and chemical nature of the interhelical interactions between the residues of transmembrane (TM) domains during different receptor activation states, something that cannot be characterized solely by visual inspection of structural information. Using the fragment molecular orbital (FMO) quantum mechanics method to analyze 35 crystal structures representing different branches of the class A GPCR family, we have identified 69 topologically equivalent TM residues that form a consensus network of 51 inter-TM interactions, providing novel results that are consistent with and help to rationalize experimental data. This discovery establishes a comprehensive picture of how defined molecular forces govern specific interhelical interactions which, in turn, support the structural stability, ligand binding, and activation of GPCRs.

Drug-target residence time, the length of time for which a small molecule stays bound to its receptor target, has increasingly become a key property for optimization in drug discovery programs. However, its in silicoprediction has proven difficult. Here we describe a method, using atomistic ensemble-based steered molecular dynamics (SMD), to observe the dissociation of ligands from their target G protein-coupled receptor in a time scale suitable for drug discovery. These dissociation simulations accurately, precisely, and reproducibly identify ligand-residue interactions and quantify the change in ligand energy values for both protein and water. The method has been applied to 17 ligands of the A2Aadenosine receptor, all with published experimental kinetic binding data. The residues that interact with the ligand as it dissociates are known experimentally to have an effect on binding affinities and residence times. There is a good correlation (R2= 0.79) between the computationally calculated change in water-ligand interaction energy and experimentally determined residence time. Our results indicate that ensemble-based SMD is a rapid, novel, and accurate semi-empirical method for the determination of drug-target relative residence time.

The accurate identification of the specific points of interaction between G protein-coupled receptor (GPCR) oligomers is essential for the design of receptor ligands targeting oligomeric receptor targets. A coarse-grained molecular dynamics computer simulation approach would provide a compelling means of identifying these specific protein–protein interactions and could be applied both for known oligomers of interest and as a high-throughput screen to identify novel oligomeric targets. However, to be effective, this in silico modeling must provide accurate, precise, and reproducible information. This has been achieved recently in numerous biological systems using an ensemble-based all-atom molecular dynamics approach. In this study, we describe an equivalent methodology for ensemble-based coarse-grained simulations. We report the performance of this method when applied to four different GPCRs known to oligomerize using error analysis to determine the ensemble size and individual replica simulation time required. Our measurements of distance between residues shown to be involved in oligomerization of the fifth transmembrane domain from the adenosine A2Areceptor are in very good agreement with the existing biophysical data and provide information about the nature of the contact interface that cannot be determined experimentally. Calculations of distance between rhodopsin, CXCR4, and β1AR transmembrane domains reported to form contact points in homodimers correlate well with the corresponding measurements obtained from experimental structural data, providing an ability to predict contact interfaces computationally. Interestingly, error analysis enables identification of noninteracting regions. Our results confirm that GPCR interactions can be reliably predicted using this novel methodology.

Purinergic signaling was first recognized in the guinea pig (Cavia porcellus) taenia coli, where relaxation of smooth muscle by nerve-released ATP may involve the activation of P2Y(1) and P2Y(11) receptors, and where transcripts for both genes have been found. A partial sequence for P2Y(11) protein was identified; the full-length P2Y(1) sequence has already been described. P2Y(1) and P2Y(11) proteins were localized by immunohistochemistry in smooth muscle cells. P2X(2) and P2X(3) proteins were also localized in motoneurons of the myenteric plexus. alphabeta-Methylene-ATP (alphabetameATP) and dibenzoyl-ATP (BzATP) evoked fast relaxations in the taenia, and they were inhibited by the P2Y(1) receptor antagonist 2'-deoxy-N(6)-methyladenosine 3',5'-bisphosphate (MRS2179). However, alphabetameATP and BzATP may stimulate neuronal P2X receptors to release ATP, which then acts on P2Y(1) receptors. In accordance, fast relaxations evoked by alphabetameATP and BzATP were inhibited by the P2X(3) and P2X(2/3) receptor antagonist 5-({[3-phenoxybenzyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl] amino} carbonyl)-1,2,4-benzene-tricarboxylic acid (A317491). When P2Y(1), P2X(3), and P2X(2/3) receptors were blocked and adenosine was removed enzymatically, alphabetameATP and BzATP evoked slow relaxations that were inhibited by Reactive Red. Fast and slow relaxations involve small and large conductance calcium-activated potassium channels; the latter are dependent on intracellular cyclic AMP levels, which altered the duration and amplitude of relaxations. alphabetameATP and BzATP were confirmed as agonists, and Reactive Red as an antagonist, of human P2Y(11) receptors. In summary, G(q)-coupled P2Y(1) receptors are involved mainly in fast relaxations, whereas G(q)and G(s)-coupled P2Y(11) receptors are involved in both fast and slow relaxations. These P2Y receptor subtypes, plus neuronal P2X receptors, may explain the phenomenon of parasympathetic inhibition first described by Langley (1898).

Jet-based technologies are increasingly being explored as potential high-throughput and high-resolution methods for the manipulation of biological materials. Previously shown to be of use in generating scaffolds from biocompatible materials, we were interested to explore the possibility of using electrospinning technology for the generation of scaffolds comprised of living cells. For this, it was necessary to identify appropriate parameters under which viable threads containing living cells could be produced. Here, we describe a method of electrospinning that can be used to deposit active biological threads and scaffolds. This has been achieved by use of a coaxial needle arrangement where a concentrated living biosuspension flows through the inner needle and a medical-grade poly(dimethylsiloxane) (PDMS) medium with high viscosity (12?500 mPa s) and low electrical conductivity (10-15S m-1) flows through the outer needle. Using this technique, we have identified the operational conditions under which the finest cell-bearing composite microthreads are formed. Collected cells that have been cultured, postelectrospinning, have been viable and show no evidence of having incurred any cellular damage during the bionanofabrication process. This study demonstrates the feasibility of using coaxial electrospinning technology for biological and biomedical applications requiring the deposition of living cells as composite microthreads for forming active biological scaffolds.

Electrospraying, or electrohydrodynamic jetting, is one of several jet-based technologies being explored to process living biological organisms. One of the key advantages of electrospraying is its ability to deposit advanced materials with high resolution that cannot be obtained with other competing technologies, such as ink-jet printing. However, to generate a controlled droplet size distribution in the micrometre range necessary for precision drop and placement of materials requires jetting in stable cone-jet mode. In this paper, we describe the experimental set-up and conditions by which electrospray jetting in stable cone-jet is achieved and use this methodology to process a highly concentrated biological suspension having 107cells ml−1, the highest cellular loading processed to this day by a jetting approach in this jet based category. The areas of study to which this technology may be applied span the physical and the life sciences.

Rat P2X(1) and P2X(2) subunits were coexpressed in defolliculated Xenopus oocytes and the resultant P2X receptors studied under voltage-clamp conditions. Extracellular ATP elicited biphasic inward currents, involving an initial rapidly inactivating (P2X(1)-like) component and a later slowly inactivating (P2X(2)-like) component. The maximum amplitude of P2X(1)-like ATP responses was increased in some cells by lowering extracellular pH (from 7.5 to 6.5), whereas P2X(2)-like responses and those of homomeric rP2X(1) and rP2X(2) receptors were not changed by this treatment. Concentration-response (C/R) curves for ATP for pH-enhanced P2X(1)-like responses were biphasic, and clearly distinct from monophasic ATP C/R curves for homomeric rP2X(1) and rP2X(2) receptors. Under acidic (pH 5.5 and 6.5) and alkaline (pH 8.5) conditions, ATP C/R curves for P2X(1)-like responses showed increases in agonist potency and efficacy, compared with data at pH 7.5, but the same was not true of homomeric rP2X(1) and rP2X(2) receptors. ATP C/R curves for P2X(2)-like responses overlay C/R curves for homomeric rP2X(2) receptors, and determinations of agonist potency and efficacy were identical for P2X(2)-like and P2X(2) responses at all pH levels tested. Our results show that P2X(1)-like responses possessed the kinetics of homomeric P2X(1) receptors but an acid sensitivity different from homomeric P2X(1) and P2X(2) receptors. In contrast, the P2X(2)-like responses exactly matched the profile expected of homomeric P2X(2) receptors. Thus, coexpression of P2X(1) and P2X(2) subunits yielded a mixed population of homomeric and heteromeric P2X receptors, with a subpopulation of novel pH-sensitive P2X receptors showing identifiably unique properties that indicated the formation of heteromeric P2X(1/2) ion channels.

Using voltage-clamp procedures on Xenopus oocytes, agonist-evoked ionic currents by P2X receptors resulting from the coexpression of P2X(2) and P2X(3) subunits were compared against the agonist responses of homomeric P2X(2) and P2X(3) receptors. With the quantity of P2X(3) mRNA kept constant and quantity of P2X(2) mRNA progressively increased, expressed P2X receptors changed from a P2X(3)-like receptor to a P2X(2)-like receptor. In all cases, however, agonist-evoked responses comprised biphasic (fast and slow) currents-the former showing the properties of P2X(3) receptors and latter consistent with the presence of P2X(2) and P2X(2/3) receptors. Using desensitization procedures, the P2X(3)-like fast current was selectively removed to allow the slow current to be studied in isolation. P2X(2/3) receptors were then characterized by slowly inactivating inward currents that were reproducible within 30 s of washout and whose pharmacological profile [selective agonists, Ap(5)A > alpha,beta-methylene ATP >> beta,gamma-methylene ATP > UTP; antagonists, TNP-ATP >> suramin > or = Reactive blue-2 (RB-2)] contrasted with the profile of P2X(2) receptors (Ap(5)A, alpha,beta-methylene ATP, beta,gamma-methylene ATP, and UTP inactive; antagonists, RB-2 > TNP-ATP > suramin). Thus, our experiments reveal that coexpression of two P2X subunits, which of themselves can generate functional homomeric receptors, results in a complex population of heterogeneous P2X receptors-in this case P2X(2), P2X(3), and P2X(2/3) receptors. Depending on the relative levels of P2X subunit coexpression, the operational profile of the resultant P2X receptors can change from one phenotype to another. This spectrum may explain the variability of agonist responses in small sensory neurons that also express P2X(2) and P2X(3) subunits in different amounts.

Responses to extracellular nucleotides (e.g., ATP, ADP, etc.) have been demonstrated in a number of embryonic cell types suggesting they may be important signalling molecules during embryonic development. Here the authors describe for the first time the expression of a G-protein–coupled receptor for extracellular ATP, chick P2Y1 (cP2Y1), during embryonic development of the chick. During the first 10 days of embryonic development, cP2Y1 is expressed in a developmentally regulated manner in the limb buds, mesonephros, brain, somites, and facial primordia, suggesting that this receptor may have a role in the development of each of these systems. Dev Dyn 1999;214:152–158. © 1999 Wiley-Liss, Inc.

AbstractVarious adenosine analogues were tested at the adenosine A2Breceptor. Agonist potencies were determined by measuring the cyclic AMP production in Chinese Hamster Ovary cells expressing human A2Breceptors. 5′-.N-Substituted carboxamidoadenosines were most potent. 5′-N-Ethylcarboxamidoadenosine (NECA) was most active with an ECso value of 3.1 μM. Other ribose modified derivatives displayed low to negligible activity. Potency was reduced by substitution on the exocyclic amino function (N6) of the purine ring system. The most active N6-substituted derivative N6-methyl-NECA was 5 fold less potent than NECA. C8-and most C2-substituted analogues were virtually inactive. 1-Deaza-analogues had a reduced potency, 3-and 7-deazaanalogues were not active.

Follicular Xenopus oocytes possess a novel receptor where both adenosine and ATP activate a cAMP-dependent, nonrectifying K+-current. Five compounds, alpha,beta-methylene ATP (alpha, beta-meATP), 8-(p-sulfophenyl)theophylline (8-SPT), theophylline, 2, 2'-pyridylisatogen tosylate (PIT) and suramin, were tested as antagonists of adenosine- and ATP-activated K+-currents. The descending order of activity (pIC50 values) against adenosine responses was: alpha,beta-meATP (6.72) = 8-SPT (6.68) > theophylline (5.32) > PIT (4.58), whereas suramin was relatively inactive. The blocking actions of alpha,beta-meATP and alkylxanthine compounds were reversible with washout, whereas blockade by PIT was irreversible. These antagonists showed similar blocking activity against ATP responses, except for PIT which was more effective at ATP responses than at adenosine responses. The selectivity of antagonists was tested against cAMP-dependent K+-currents evoked by forskolin and follicle-stimulating hormone (FSH). 8-SPT and theophylline did not inhibit but instead augmented forskolin and FSH responses; this augmentation may be caused by inhibition of phosphodiesterase activity inside follicle cells. On the other hand, alpha,beta-MeATP and PIT inhibited forskolin and FSH responses; both compounds apparently are nonselective antagonists. Thus, only alkylxanthine derivatives (8-SPT and theophylline) were selective antagonists of the novel adenosine/ATP receptor in Xenopus oocytes, whereas alpha,beta-meATP and PIT were nonselective in their blocking actions and suramin was relatively inactive.

The gene for the stimulatory G protein-coupled human A2a adenosine receptor was isolated and sequence analysis revealed two exons that are interrupted by an intron of approximately 6.4 kb. An intron is located in the same region in the human A1 and A2b adenosine receptor genes. Comparison of the A2a genomic and cDNA sequences reveals two nucleotide differences in the coding region and the presence of an aberrant sequence in the 5′208 base pairs of the A2a cDNA including a polymorphism in the third base of codon Tyr-361 and a Gly codon which was always detected at residue 392, indicating that the Arg codon present in the cDNA may be an artifact. Fluorescentin situhybridization and PCR analysis of human-hamster hybrid cell panels shows that the A2a receptor gene is localized to chromosome 22q11.2. This is in contrast with previous reports (subsequently retracted) which mapped the A2a gene to chromosome 11q11–13.

P2 receptors have been identified in rat kidney by autoradiography, using the radioligand [3H]α,β-methylene ATP, and by immunohistochemistry, using a polyclonal antibody to the P2X1purinoceptor. They have been localized to the vascular smooth muscle of intrarenal arteries, including arcuate and interlobular arteries, and afferent arterioles, but not glomeruli, postglomerular efferent arterioles, or renal tubules. We conclude that at least some of the P2 receptors present on vascular smooth muscle are of the P2X1subtype. The functional significance of these findings in the vascular control of the kidney is discussed.

We apply the hit-to-lead ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) and lead-optimization TIES (thermodynamic integration with enhanced sampling) methods to compute the binding free energies of a series of ligands at the A1and A2Aadenosine receptors, members of a subclass of the GPCR (G protein-coupled receptor) superfamily. Our predicted binding free energies, calculated using ESMACS, show a good correlation with previously reported experimental values of the ligands studied. Relative binding free energies, calculated using TIES, accurately predict experimentally determined values within a mean absolute error of approximately 1 kcal mol−1. Our methodology may be applied widely within the GPCR superfamily and to other small molecule–receptor protein systems.

The identification of strategies by which to increase the representation of women and increase diversity in STEM fields (science, technology, engineering and mathematics), including medicine, has been a pressing matter for global agencies including the European Commission, UNESCO and numerous international scientific societies. In my role as UCL training lead for CompBioMed, a European Commission Horizon 2020-funded Centre of Excellence in Computational Biomedicine (compbiomed.eu), and as Head of Teaching for Molecular Biosciences at UCL from 2010 to 2019, I have integrated research and teaching to lead the development of high-performance computing (HPC)-based education targeting medical students and undergraduate students studying biosciences in a way that is explicitly integrated into the existing university curriculum as a credit-bearing module. One version of the credit-bearing module has been specifically designed for medical students in their pre-clinical years of study and one of the unique features of the course is the integration of clinical and computational aspects, with students obtaining and processing clinical samples and then interrogating the results computationally using code that was ported to HPC at CompBioMed's HPC Facility core partners (EPCC (UK), SURFsara (The Netherlands) and the Barcelona Supercomputing Centre (Spain)). Another version of the credit-bearing module has, over the course of this project, evolved into a replacement for the third year research project course for undergraduate biochemistry, biotechnology and molecular biology students, providing students with the opportunity to design and complete an entire specialist research project from the formulation of experimental hypotheses to the investigation of these hypotheses in a way that involves the integration of experimental and HPC-based computational methodologies. Since 2017–2018, these UCL modules have been successfully delivered to over 350 students—a cohort with a demographic of greater than 50% female. CompBioMed's experience with these two university modules has enabled us to distil our methodology into an educational template that can be delivered at other universities in Europe and worldwide. This educational approach to training enables new communities of practice to effectively engage with HPC and reveals a means by which to improve the underrepresentation of women in supercomputing.

A recent genome-wide association study identified a locus within natriuretic peptide receptor C (NPR3) gene to be significantly associated with blood pressure (BP). We aim to functionally characterise the effect of BP- associated SNPs (single nucleotide polymorphisms) at the NPR3 gene locus in the context of BP regulatory pathways.

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