New Binding Sites, New Opportunities for GPCR Drug Discovery.

Many central biological events rely on protein-ligand interactions. The identification and characterization of protein-binding sites for ligands are crucial for the understanding of functions of both endogenous ligands and synthetic drug molecules. G protein-coupled receptors (GPCRs) typically detect extracellular signal molecules on the cell surface and transfer these chemical signals across the membrane, inducing downstream cellular responses via G proteins or ß-arrestin. GPCRs mediate many central physiological processes, making them important targets for modern drug discovery. Here, we focus on the most recent breakthroughs in finding new binding sites and binding modes of GPCRs and their potentials for the development of new medicines.

Translocation Mechanism of Allosteric Sodium Ions in ß2-Adrenoceptor.

The allosteric modulation of G-protein-coupled receptors (GPCRs) by sodium ions has received significant attention as the crystal structures of several receptors show the binding of sodium ions (Na+) at the conserved D2.50. Theoretical studies have shown that extracellular Na+ would enter the allosteric D2.50 via the orthosteric site. However, it remains unclear how the bound allosteric Na+ would leave the GPCRs. In this study, we performed molecular dynamics (MD) simulations to illustrate the energy barriers of Na+ transfer through the transmembrane helix bundle of ß2AR. In contrast to the postulations from other GPCRs, the translocation of this allosteric Na+ into the intracellular side is found to be significantly difficult. Hence, the translocation direction could be receptor-specific.

Accurate Physical Property Predictions via Deep Learning.

Neural networks and deep learning have been successfully applied to tackle problems in drug discovery with increasing accuracy over time. There are still many challenges and opportunities to improve molecular property predictions with satisfactory accuracy even further. Here, we proposed a deep-learning architecture model, namely Bidirectional long short-term memory with Channel and Spatial Attention network (BCSA), of which the training process is fully data-driven and end to end. It is based on data augmentation and SMILES tokenization technology without relying on auxiliary knowledge, such as complex spatial structure. In addition, our model takes the advantages of the long- and short-term memory network (LSTM) in sequence processing. The embedded channel and spatial attention modules in turn specifically identify the prime factors in the SMILES sequence for predicting properties. The model was further improved by Bayesian optimization. In this work, we demonstrate that the trained BSCA model is capable of predicting aqueous solubility. Furthermore, our proposed method shows noticeable superiorities and competitiveness in predicting oil-water partition coefficient, when compared with state-of-the-art graphs models, including graph convoluted network (GCN), message-passing neural network (MPNN), and AttentiveFP.

Effect of SARS and COVID-19 outbreaks on urology practice and training.

INTRODUCTION: The objective was to investigate the changes in urology practice during coronavirus disease 2019 (COVID-19) pandemic with a perspective from our experience with severe acute respiratory syndrome (SARS) in 2003. METHODS: Institutional data from all urology centres in the Hong Kong public sector during the COVID-19 pandemic (1 Feb 2020-31 Mar 2020) and a non-COVID-19 control period (1 Feb 2019-31 Mar 2019) were acquired. An online anonymous questionnaire was used to gauge the impact of COVID-19 on resident training. The clinical output of tertiary centres was compared with data from the SARS period. RESULTS: The numbers of operating sessions, clinic attendance, cystoscopy sessions, prostate biopsy, and shockwave lithotripsy sessions were reduced by 40.5%, 28.5%, 49.6%, 44.8%, and 38.5%, respectively, across all the centres reviewed. The mean numbers of operating sessions before and during the COVID-19 pandemic were 85.1±30.3 and 50.6±25.7, respectively (P=0.005). All centres gave priority to cancer-related surgeries. Benign prostatic hyperplasia-related surgery (39.1%) and ureteric stone surgery (25.5%) were the most commonly delayed surgeries. The degree of reduction in urology services was less than that during SARS (47.2%, 55.3%, and 70.5% for operating sessions, cystoscopy, and biopsy, respectively). The mean numbers of operations performed by residents before and during the COVID-19 pandemic were 75.4±48.0 and 34.9±17.2, respectively (P=0.002). CONCLUSION: A comprehensive review of urology practice during the COVID-19 pandemic revealed changes in every aspect of practice.

MolADI: A Web Server for Automatic Analysis of Protein-Small Molecule Dynamic Interactions.

Protein-ligand interaction analysis is important for drug discovery and rational protein design. The existing online tools adopt only a single conformation of the complex structure for calculating and displaying the interactions, whereas both protein residues and ligand molecules are flexible to some extent. The interactions evolved with time in the trajectories are of greater interest. MolADI is a user-friendly online tool which analyzes the protein-ligand interactions in detail for either a single structure or a trajectory. Interactions can be viewed easily with both 2D graphs and 3D representations. MolADI is available as a web application.

Emergency attendances and hospitalisations for complications after transrectal ultrasound-guided prostate biopsies: a five-year retrospective multicentre study.

INTRODUCTION: Transrectal ultrasound-guided (TRUS) prostate biopsy is an established procedure for diagnosis of prostate cancer. Complications after TRUS biopsy are not well reported in Hong Kong. This study evaluated the 5-year incidences of TRUS biopsy complications and potential risk factors for those complications. METHODS: This was a retrospective review of biopsies performed from 2013 to 2017 in two local hospitals, using data retrieved from electronic medical records. The primary outcome was the occurrence of complications requiring either emergency attendances or hospitalisations within 30 days after biopsy. Potential risk factors were examined using multiple logistic regression analysis. RESULTS: In total, 1699 men were included (mean age ± standard deviation: 67 ± 7 years; median prostate-specific antigen level: 7.9 µg/L [interquartile range, 5.5-12.6 µg/L]); 4.3% had pre-biopsy bacteriuria. Overall, 5.7% and 3.8% of post-biopsy complications required emergency attendances and hospitalisations, respectively. Gross haematuria and rectal bleeding requiring emergency attendances developed in 2.1% and 0.4% of men; 0.8% and 0.4% required hospitalisations. Furthermore, 1.5% of men developed acute urinary retention requiring hospitalisations; 1.9% and 1.2% had post-biopsy infections requiring emergency attendances and hospitalisations, respectively, and 0.9% had urosepsis requiring hospitalisations. Prostate volume >48 cc was associated with an increased risk of post-biopsy retention (odds ratio 2.75, 95% confidence interval: 1.23-4.17). CONCLUSIONS: The rate of overall complications after TRUS biopsy was low. The most common complications requiring emergency attendances and hospitalisations were gross haematuria and acute urinary retention, respectively. Prostate volume >48 cc increased the risk of post-biopsy urinary retention.

Structural and Computational Insight into the Catalytic Mechanism of Limonene Epoxide Hydrolase Mutants in Stereoselective Transformations.

Directed evolution of limonene epoxide hydrolase (LEH), which catalyzes the hydrolytic desymmetrization reactions of cyclopentene oxide and cyclohexene oxide, results in (R,R)- and (S,S)-selective mutants. Their crystal structures combined with extensive theoretical computations shed light on the mechanistic intricacies of this widely used enzyme. From the computed activation energies of various pathways, we discover the underlying stereochemistry for favorable reactions. Surprisingly, some of the most enantioselective mutants that rapidly convert cyclohexene oxide do not catalyze the analogous transformation of the structurally similar cyclopentene oxide, as shown by additional X-ray structures of the variants harboring this slightly smaller substrate. We explain this puzzling observation on the basis of computational calculations which reveal a disrupted alignment between nucleophilic water and cyclopentene oxide due to the pronounced flexibility of the binding pocket. In contrast, in the stereoselective reactions of cyclohexene oxide, reactive conformations are easily reached. The unique combination of structural and computational data allows insight into mechanistic details of this epoxide hydrolase and provides guidance for future protein engineering in reactions of structurally different substrates.

Manipulation of polarization and spatial properties of light beams with chiral metafilms.

Two-dimensional lattices of chiral nanoholes in a plasmonic film with lattice constants being slightly larger than light wavelength are proposed for effective control of polarization and spatial properties of light beams. Effective polarization conversion and strong circular dichroism in non-zero diffraction orders in these chiral metafilms are demonstrated by electromagnetic simulations. These interesting effects are found to result from interplay between radiation pattern of single chiral nanohole and diffraction pattern of the planar lattice, and can be manipulated by varying wavelength and polarization of incoming light as well as period of metastructure and refractive indexes of substrate and overlayer. Therefore, this work offers a novel paradigm for developing planar chiral metafilm-based optical devices with controllable polarization state, spatial orientation and intensity of outgoing light.

The Molecular Mechanism of P2Y1 Receptor Activation.

Human purinergic G protein-coupled receptor P2Y1 (P2Y1 R) is activated by adenosine 5'-diphosphate (ADP) to induce platelet activation and thereby serves as an important antithrombotic drug target. Crystal structures of P2Y1 R revealed that one ligand (MRS2500) binds to the extracellular vestibule of this GPCR, whereas another (BPTU) occupies the surface between transmembrane (TM) helices TM2 and TM3. We introduced a total of 20 µs all-atom long-timescale molecular dynamic (MD) simulations to inquire why two molecules in completely different locations both serve as antagonists while ADP activates the receptor. Our results indicate that BPTU acts as an antagonist by stabilizing extracellular helix bundles leading to an increase of the lipid order, whereas MRS2500 blocks signaling by occupying the ligand binding site. Both antagonists stabilize an ionic lock within the receptor. However, binding of ADP breaks this ionic lock, forming a continuous water channel that leads to P2Y1 R activation.

Modeling of Olfactory Receptors.

Olfactory receptors (ORs) form the largest subfamily within class A G protein-coupled receptors (GPCRs). No experimental structural data of any OR is available to date. Homology modeling has become a popular strategy to propose plausible OR models, in order to study the structure-function relationships of the receptors and to aid the discovery and development of ligands capable of modulating receptor activity. In this chapter, we provide a general guideline for OR structure construction, including the collection of candidate templates, structure-based sequence alignment, 3D structure construction, ligand docking, and molecular dynamic simulation.

Two Novel Metformin Carboxylate Salts and the Accidental Discovery of Two 1,3,5-Triazine Antihyperglycemic Agent.

Metformin (MET), commonly marketed as a hydrochloride salt (MET-HCl) for better pharmacokinetic profile over the free base, would release a high concentration of chloride ions and cause adverse gastrointestinal effects. The preparation of chloride-free MET salts could potentially circumvent this issue. In this study, seven carboxylic acids (formic acid, acetic acid, malonic acid, succinic acid, fumaric acid, cinnamic acid, and acetylsalicylic acid) were used for preparing MET carboxylate salts. When compared with MET-HCl, all MET salts/salt hydrates show lower dissolution rates in pH 6.8 phosphate buffer. However, the cinnamic acid and acetylsalicylic acid show significantly higher dissolution rates in the forms of MET salt/salt hydrate. In the permeability test, the permeability of the MET in all of the salts was not improved. However, the permeability of cinnamic acid in the MET cinnamate is reduced, and the permeability of acetylsalicylic acid in the MET acetylsalicylate is increased. Meanwhile, at a higher crystallization temperature, the acetone solvent and a hydrolyzed product of acetylsalicylic acid react with MET respectively, leading to two unexpected 1,3,5-triazine derivatives. The results of in vitro bioactivity assays indicate that one of the triazine molecules promote glucose consumption more effectively than MET-HCl, and had relatively weak lactate production ability at low concentration. This glucose metabolism regulating compound may serve as a novel lead antihyperglycemic agent for further optimization.

Predictability of the polymorphs of small organic compounds: crystal structure predictions of four benchmark blind test molecules.

Predicting the crystal structure of an organic molecule from first principles has been a major challenge in physical chemistry. Recently, the application of Density Functional Theory including a dispersive energy correction (the DFT(d) method) has been shown to be a reliable method for predicting experimental structures based purely on their ranking according to lattice energy. Further validation results of the application of the DFT(d) method to four organic molecules are presented here. The compounds were targets (labelled molecule II, VI, VII and XI) in previous blind tests of crystal structure prediction, and their structures proved difficult to predict. However, this study shows that the DFT(d) approach is capable of predicting the solid state structures of these small molecules. For molecule VII, the most stable (rank 1) predicted crystal structure corresponds to the experimentally observed structure. For molecule VI, the rank 1, 2 and 3 predicted structures correspond to the three experimental polymorphs, forms I, III and II, respectively. For molecules II and XI, their rank 1 predicted structures are energetically more stable than those corresponding to the experimental crystal structures, and were not found amongst the structures submitted by the participants in the blind tests. The rank 1 structure of molecule II is predicted to exist under high pressure, whilst the rank 1 structure predicted for molecule XI has the same space group and hydrogen bonding pattern as observed in the crystal of 1-amino-1-methyl-cyclopropane, which is structurally related to molecule XI. The experimental crystal structure of molecule II corresponds to the rank 4 prediction, 0.8 kJ mol(-1) above the global minimum structure, and the experimental structure of molecule XI corresponds to the rank 2 prediction, 0.4 kJ mol(-1) above the global minimum.

Phase solubility diagrams and energy surface calculations support the solubility enhancement with low hygroscopicity of Bergenin: 4-Aminobenzamide (1: 1) cocrystal.

Herein, we reported a new bergenin: 4-aminobenzamide (BGN-4AM) cocrystal with significantly enhanced solubility and low hygroscopicity probed from two aspects such as phase solubility diagrams and theoretical calculations. Compared with anhydrous BGN, BGN-4AM solubilities in water and different buffer solutions (pH = 1.2, 4.5, 6.8) increase significantly. It is noted that BGN-4AM solubility in pH = 6.8 buffer solution presents 32.7 times higher than anhydrous BGN. Interestingly, BGN-4AM (0.31 ± 0.07%) showcases lower hygroscopicity than anhydrous BGN (9.31 ± 0.16%). The predicted and experimental solubilities agree with each other when considering solubility product (Ksp) and solution binding constant (K11) in phase solubility diagrams, indicating the solution complexes formation occurs. Further crystal surface-water interactions and Bravais, Friedel, Donnay-Harker (BFDH) analyses based on Density Functional Theory with dispersion correction (DFT-d) methods support the enhanced solubility. The water probe demonstrates an average interaction energy of -6.48 kcal/mol on the 002 plane of BGN-4AM, and only -5.47 kcal/mol on the 011 plane of BGN monohydrate. The lower lattice energy of BGN-4AM guarantees its lower hygroscopicity than BGN monohydrate. BGN-4AM with enhanced solubility and low hygroscopicity can be a potential candidate for further formulation development.

Suppressor of cytokine signaling 1 gene expression and polymorphisms in systemic lupus erythematosus.

With the aim of investigating the role of suppressor of cytokine signaling 1 (SOCS1) in the pathogenesis of systemic lupus erythematosus, 107 patients with systemic lupus erythematosus, 101 healthy controls, and 151 patients with ankylosing spondylitis were enrolled in this study. SOCS1 mRNA level was measured by the method of quantitative real-time polymerase chain reaction. SOCS1 polymorphisms were detected by the polymerase chain reaction/restriction fragment length polymorphisms method. Systemic lupus erythematosus disease activity was evaluated with the SLEDAI. This study showed that the SOCS1 mRNA expression was significantly higher in the patients with systemic lupus erythematosus than in the healthy controls (p = 0.0014). Patients with active systemic lupus erythematosus had a higher expression of SOCS1 mRNA than the patients with inactive systemic lupus erythematosus (p = 0.035). There was no significant difference in the frequencies of the SOCS1-1478CA/del polymorphisms among the patients with systemic lupus erythematosus, healthy controls, and patients with ankylosing spondylitis. The genotype frequency of the SOCS1-1478 polymorphisms in the dominant model (CA/del+del/del versus CA/CA) was significantly decreased in the patients with thrombocytopenia compared with those without thrombocytopenia (p(c) = 0.035). Moreover, the allele frequency of SOCS1-1478del was also significantly lower in the patients with thrombocytopenia than in those without thrombocytopenia (p( c) = 0.02). In conclusion, this study demonstrated that the expression of SOCS1 mRNA was significantly increased in patients with systemic lupus erythematosus. Moreover, SOCS1 mRNA levels in patients with active systemic lupus erythematosus were significantly higher than those in the inactive patients. We also found that the systemic lupus erythematosus patients with thrombocytopenia have a lower frequency of SOCS1-1478del compared with patients without thrombocytopenia.

A sperm GPI-anchored protein elicits sperm-cumulus cross-talk leading to the acrosome reaction.

The acrosome reaction has long been thought to be induced by the zona pellucida. Here we report the identification and function of a novel human sperm glycosylphosphatidylinositol (GPI)-anchored membrane protein, NYD-SP8. The release of the protein during sperm-egg interaction and its binding to the cumulus, the first layer of egg investment, elicits cross-talk between the gametes and produces calcium dependant release of progesterone, which lead to the acrosome reaction. An in vivo mouse model of NYD-SP8 immunization is also established showing a reduced fertility rate. Thus, contrary to accepted dogma, our study demonstrates for the first time that, prior to reaching the zona pellucida, sperm may release a surface protein that acts on the cumulus cells leading to the acrosome reaction, which may be important for determining the outcome of fertilization.

Seroprevalence of Toxocara canis infection among children in Swaziland, southern Africa.

As there appeared to be no data available on Toxocara canis infection in the children of Swaziland, a serological survey of T. canis infection was recently conducted among 92 children aged 3-12 years from rural slums in the low- and middle-veld. A child was considered seropositive if, in western blots based on the excretory-secretory antigens of larval T. canis, his or her serum gave a positive result when diluted 1 : 64. Forty-one (44.6%) of the children were found seropositive. There were no statistically significant differences in seroprevalence between the 49 boys and 43 girls investigated (46.9% v. 41.8%) or between the eight subjects aged 12 years and the 47 aged < or = 5 years (62.5% v. 38.3%); the corresponding odds ratios were 0.81 (95% confidence interval=0.36-1.86; P=0.62) and 2.69 (95% confidence interval=0.57-12.62; P=0.20), respectively. The 66 subjects from the middleveld were, however, significantly more likely to be seropositive than the 26 subjects from the lowveld (54.5% v. 19.2%; odds ratio=5.04, with a 95% confidence interval of 1.70-14.98; P<0.01). It seems likely that T. canis infection is common among the children who live in slums in Swaziland, particularly in the country's middleveld, probably as the result of poor hygiene and poor sanitation.

Enhancing the Signaling of GPCRs via Orthosteric Ions.

G protein-coupled receptors play essential roles in cellular processes such as neuronal signaling, vision, olfaction, tasting, and metabolism. As GPCRs are the most important drug targets, understanding their interactions with ligands is of utmost importance for discovering related new medicines. In many GPCRs, an allosteric sodium ion next to the highly conserved residue D2.50 has been proposed to stabilize the inactive receptor state by mediating interactions between transmembrane helices. Here, we probed the existence of internal and functionally important sodium ions in the dopamine D2 receptor, using molecular dynamics simulations. Besides a new sodium ion at the allosteric ligand binding site, we discovered an additional sodium ion, located close to the orthosteric ligand binding site. Through cell-based activation assays, the signaling of D2 receptor with site-specific mutations was tested against a series of chemically modified agonists. We concluded an important structural role of this newly discovered orthosteric sodium ion in modulating the receptor signaling: It enables the coordination of a polar residue in the ligand binding site with an appropriately designed agonist molecule. An identical interaction was also observed in a recently released high-resolution crystal structure of mu-opioid receptor, which was reresolved in this work. Probably because of similar interactions, various metal ions have been found to increase the signaling of many other GPCRs. This unique principle and strategy could be used to optimize the drug activity of GPCR. Our findings open a new mechanistic opportunity of GPCR signaling and help design the next generation of drugs targeting GPCRs.