Occupancy Of Binding Sites Research Articles

A new perspective on microRNA-guided gene regulation specificity, and its potential generalization to transcription factors and RNA-binding proteins.

Our conception of gene regulation specificity has undergone profound changes over the last 20years. Previously, regulators were considered to control few genes, recognized with exquisite specificity by a 'lock and key' mechanism. However, recentlygenome-wide exploration of regulator binding site occupancy (whether on DNA or RNA targets) revealed extensive lists of molecular targets for every studied regulator. Such poor biochemical specificity suggested that each regulator controls many genes, collectively contributing to biological phenotypes. Here, I propose a third model, whereby regulators' biological specificity is only partially due to 'lock and key' biochemistry. Rather, regulators affect many genes at the microscopic scale, but biological consequences for most interactions are attenuated at the mesoscopic scale: only a few regulatory events propagate from microscopic to macroscopic scale;others are made inconsequential by homeostatic mechanisms. This model is well supported by the microRNA literature, and data suggest that it extends to other regulators. It reconciles contradicting observations from biochemistry and comparative genomics on one hand and in vivo genetics on the other hand, but this conceptual unification is obscured by common misconceptions and counter-intuitive modes of graphical display. Profound understanding of gene regulation requires conceptual clarification, and better suited statistical analyses and graphical representation.

A lactate-dependent shift of glycolysis mediates synaptic and cognitive processes in male mice

Astrocytes control brain activity via both metabolic processes and gliotransmission, but the physiological links between these functions are scantly known. Here we show that endogenous activation of astrocyte type-1 cannabinoid (CB1) receptors determines a shift of glycolysis towards the lactate-dependent production of d-serine, thereby gating synaptic and cognitive functions in male mice. Mutant mice lacking the CB1 receptor gene in astrocytes (GFAP-CB1-KO) are impaired in novel object recognition (NOR) memory. This phenotype is rescued by the gliotransmitter d-serine, by its precursor l-serine, and also by lactate and 3,5-DHBA, an agonist of the lactate receptor HCAR1. Such lactate-dependent effect is abolished when the astrocyte-specific phosphorylated-pathway (PP), which diverts glycolysis towards l-serine synthesis, is blocked. Consistently, lactate and 3,5-DHBA promoted the co-agonist binding site occupancy of CA1 post-synaptic NMDA receptors in hippocampal slices in a PP-dependent manner. Thus, a tight cross-talk between astrocytic energy metabolism and gliotransmission determines synaptic and cognitive processes.

Insight into the K channel's selectivity from binding of K+, Na+ and water to N-methylacetamide.

In potassium channels that conduct K+ selectively over Na+, which sites are occupied by K+ or water and the mechanism of selectivity are unresolved questions. The combination of the energetics and the constraints imposed by the protein structure yield the selective permeation and occupancy. To gain insight into the combination of structure and energetics, we performed density functional theory (DFT) calculations of multiple N-methyl acetamide (NMA) ligands binding to K+ and Na+, relative to hydrated K+ and Na+. NMA is an analogue of the amino acid backbone and provides the carbonyl binding to the ions that occurs in most binding sites of the K+ channel. Unconstrained optimal structures are obtained through geometry optimization calculations of the NMA ligand binding. The complexes formed by 8 NMA binding to the cations have the O atoms positioned in nearly identical locations as the O atoms in the selectivity filter. The transfer free energies between bulk water and K+ or Na+ bound to 8 NMA are almost identical, implying there is no selectivity by a single site. For water optimized with 8 NMA, binding is weak and O atoms are not positioned as in the K+ channel selectivity filter, suggesting that the ions are much more favored than water. Optimal structures of 8 NMA binding with two cations (K+ or Na+) are stable and have lower binding free energy than the optimal structures with just one cation. However, in the Na+ case, the optimal structure deforms and does not match the K+ channel; that is, two bound Na+ are destabilizing. In contrast, the two K+ structure is stabilized and the selectivity free energy favors K+. Overall, this study shows that binding site occupancy and the mechanism for K+ selectivity involves multiple K+ binding in multiple neighboring layers or sites of the K+ channel selectivity filter.

Probing the mechanism by which the retinal G protein transducin activates its biological effector PDE6

Phototransduction in retinal rods occurs when the G protein-coupled photoreceptor rhodopsin triggers the activation of phosphodiesterase 6 (PDE6) by GTP-bound alpha subunits of the G protein transducin (GαT). Recently, we presented a cryo-EM structure for a complex between two GTP-bound recombinant GαT subunits and native PDE6, that included a bivalent antibody bound to the C-terminal ends of GαT and the inhibitor vardenafil occupying the active sites on the PDEα and PDEβ subunits. We proposed GαT-activated PDE6 by inducing a striking reorientation of the PDEγ subunits away from the catalytic sites. However, questions remained including whether in the absence of the antibody GαT binds to PDE6 in a similar manner as observed when the antibody is present, does GαT activate PDE6 by enabling the substrate cGMP to access the catalytic sites, and how does the lipid membrane enhance PDE6 activation? Here, we demonstrate that 2:1GαT-PDE6 complexes form with either recombinant or retinal GαT in the absence of the GαT antibody. We show that GαT binding is not necessary for cGMP nor competitive inhibitors to access the active sites; instead, occupancy of the substrate binding sites enables GαT to bind and reposition the PDE6γ subunits to promote catalytic activity. Moreover, we demonstrate by reconstituting GαT-stimulated PDE6 activity in lipid bilayer nanodiscs that the membrane-induced enhancement results from an increase in the apparent binding affinity of GαT for PDE6. These findings provide new insights into how the retinal G protein stimulates rapid catalytic turnover by PDE6 required for dim light vision.

Acoramidis produces near-complete TTR stabilization in blood samples from patients with variant transthyretin amyloidosis that is greater than that achieved with tafamidis

Abstract Introduction Transthyretin (TTR) amyloidosis (ATTR) is a progressive, fatal disease caused by destabilizing TTR variants (TTRv) and age-related factors. Dissociation of tetrameric TTR initiates protein misfolding, aggregation, and tissue deposition which constitutes the mechanism of disease. More destabilizing variants drive more severe clinical phenotypes. TTR stabilizers have demonstrated clinical benefits for neuropathic and cardiovascular outcomes correlated with the extent of TTR stabilization. Acoramidis (AG10) is a novel TTR stabilizer in development for the treatment of TTR amyloid cardiomyopathy. Hypothesis: Acoramidis achieves near-complete in vitro TTR stabilization, exceeding levels achieved with clinically relevant concentrations of tafamidis (a TTR stabilizer in clinical use), when added to blood samples from ATTRv patients across a spectrum of destabilizing TTR variants. Methods Two established assays assessed TTR stabilization: fluorescent probe exclusion (FPE; measures binding site occupancy), and Western blot (WB; quantifies tetrameric TTR persistence under conditions of accelerated dissociation). Over 60 individual patient samples representing 18 unique TTRv across a spectrum of intrinsic instability and clinical phenotypes were assayed. Acoramidis was added at its target clinical trough concentration (10 µM) and compared to tafamidis added at its clinical peak (26 µM) and trough (16 µM) concentrations reported for its maximal approved dose. Results Acoramidis bound serum TTR to a greater extent (103 ± 13%) than either peak (87 ± 14%) or trough (71 ± 14%) concentrations of tafamidis. WB assays showed that addition of acoramidis resulted in significantly greater and more durable TTR stabilization (93 ± 14%) than adding tafamidis (peak: 49 ± 14%, trough: 36 ± 13%) in all paired individual patient plasma samples tested (Figure 1, p < 0.0001). Rare variants (A97S, D38A, F64L, L58H, P24S, Y114C) also demonstrated near complete stabilization upon in vitro addition of acoramidis. Conclusions At its target therapeutic trough concentration, acoramidis achieved near-complete TTR stabilization across 18 unique genotypes; in the subset of paired samples, the stabilization was significantly greater for acoramidis than for tafamidis even at its peak clinical concentration. This observation held across a range of destabilizing mutations, including a∼2-fold greater stabilization than tafamidis for the prevalent cardiomyopathic V122I variant. Based on the mechanism of disease and the association between TTR destabilization and severity of clinical outcomes, these data suggest that acoramidis has the potential to be a clinically differentiated and efficacious treatment option for patients with ATTRv, independent of variant genotype.

An Insight into Anion Extraction by Amphiphiles: Hydrophobic Microenvironments as a Requirement for the Extractant Selectivity.

Coupling of electron-deficient urea units with aliphatic chains gives rise to amphiphilic compounds that bind to phosphate and benzoate anions in the hydrogen bonding competitive solvent (DMSO) with KAss = 6 580 M-1 and KAss = 4 100 M-1, respectively. The anchoring of these receptor moieties to the dendritic support does not result in a loss of anion binding and enables new applications. Due to the formation of a microenvironment in the dendrimer, the high selectivity of the prepared compound toward benzoate is maintained even in the presence of aqueous media during extraction experiments. In the presence of binding sites at 5 mM concentration, the amount of benzoate corresponding to the full binding site occupancy is transferred into the chloroform phase from its 10 mM aqueous solution. A thorough investigation of the extraction behavior of the dendrimer reported here, supported by a series of molecular dynamics simulations, provides new insight into the fundamental principles of extraction of inorganic anions by amphiphiles.

A Fluorogenic Disaccharide Substrate for α-Mannosidases Enables High-Throughput Screening and Identification of an Inhibitor of the GH92 Virulence Factor from Streptococcus pneumoniae.

Trimming of host glycans is a mechanism that is broadly employed by both commensal and pathogenic microflora to enable colonization. Host glycan trimming by the opportunistic Gram-positive bacterium Streptococcus pneumoniae has been demonstrated to be an important mechanism of virulence. While S. pneumoniae employs a multitude of glycan processing enzymes, the exo-mannosidase SpGH92 has been shown to be an important virulence factor. Accordingly, SpGH92 is hypothesized to be a target for much-needed new treatments of S. pneumoniae infection. Here we report the synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→2)-β-d-mannopyranoside (Manα1,2Manβ-4MU) as a fluorogenic disaccharide substrate and development of an assay for SpGH92 that overcomes its requirement for +1 binding site occupancy. We miniaturize our in vitro assay and apply it to a high-throughput screen of >65 000 compounds, identifying a single inhibitory chemotype, LIPS-343. We further show that Manα1,2Manβ-4MU is also a substrate of the human Golgi-localized α-mannosidase MAN1A1, suggesting that this substrate should be useful for assessing the activity of this and other mammalian α-mannosidases.

Navigating the complex landscape of benzodiazepine- and Z-drug diversity: insights from comprehensive FDA adverse event reporting system analysis and beyond.

Medications which target benzodiazepine (BZD) binding sites of GABAA receptors (GABAARs) have been in widespread use since the nineteen-sixties. They carry labels as anxiolytics, hypnotics or antiepileptics. All benzodiazepines and several nonbenzodiazepine Z-drugs share high affinity binding sites on certain subtypes of GABAA receptors, from which they can be displaced by the clinically used antagonist flumazenil. Additional binding sites exist and overlap in part with sites used by some general anaesthetics and barbiturates. Despite substantial preclinical efforts, it remains unclear which receptor subtypes and ligand features mediate individual drug effects. There is a paucity of literature comparing clinically observed adverse effect liabilities across substances in methodologically coherent ways. In order to examine heterogeneity in clinical outcome, we screened the publicly available U.S. FDA adverse event reporting system (FAERS) database for reports of individual compounds and analyzed them for each sex individually with the use of disproportionality analysis. The complementary use of physico-chemical descriptors provides a molecular basis for the analysis of clinical observations of wanted and unwanted drug effects. We found a multifaceted FAERS picture, and suggest that more thorough clinical and pharmacoepidemiologic investigations of the heterogenous side effect profiles for benzodiazepines and Z-drugs are needed. This may lead to more differentiated safety profiles and prescription practice for particular compounds, which in turn could potentially ease side effect burden in everyday clinical practice considerably. From both preclinical literature and pharmacovigilance data, there is converging evidence that this very large class of psychoactive molecules displays a broad range of distinctive unwanted effect profiles - too broad to be explained by the four canonical, so-called "diazepam-sensitive high-affinity interaction sites". The substance-specific signatures of compound effects may partly be mediated by phenomena such as occupancy of additional binding sites, and/or synergistic interactions with endogenous substances like steroids and endocannabinoids. These in turn drive the wanted and unwanted effects and sex differences of individual compounds.

A novel approach to dissect ions occupancy and dissociation constant of individualbinding sites within the K+-channel selectivity filter.

K+-channels permeate Rb+ as well as they do K+, although with a lower single channel conductance. KcsA crystal structure displays four K+ bound to its selectivity filter (SF); however, when crystallized in Rb+, ions were only found at the binding sites 1, 3, and 4. This observation explains the lower single conductance of K+-channels in rubidium. We reported a KcsA mutant (G77A) that stabilized the SF in the 2-4 ion bound configuration with K+ ions coordinated in the 2nd and 4th binding sites. Crystallizing the KcsA-G77A structure in rubidium interestingly showed Rb+ ions bound to the 1st and 4th binding sites. Like in the wild-type channel, the 2nd binding site was uncapable of binding rubidium. Therefore, we used the G77A to measure the 1st binding site dissociation constant for Rb+ by Isothermal Titration Calorimetry (ITC). Previously, computational studies have suggested that: 1) the SF's 4th ion binding site can be occupied by K+ or Na+ and hence it should not contribute to the SF's K+ binding affinity and 2) the 2nd binding site is responsible for the K+ binding affinity displayed by K+-channels SF. In here, we are showing that in K+-channels the 2nd binding site is indeed responsible for K+ binding affinity while the 1st site is responsible for the Rb+ ion binding affinity, since the G77A mutant (a simplified structural model for the SF) showed a wild-type like Rb+ dissociation constant. Therefore, we propose that in K+-channels the 1st ion binding site is responsible for the high rubidium over sodium selectivity shown by KcsA and potentially other potassium channels. Our advances in applying this approach for the alkali metal ion series will be presented.

Cooperativity of myosin II motors in the non-regulated and regulated thin filaments investigated with high-speed AFM.

Skeletal myosins II are non-processive molecular motors that work in ensembles to produce muscle contraction while binding to the actin filament. Although the molecular properties of myosin II are well known, there is still debate about the collective work of the motors: is there cooperativity between myosin motors while binding to the actin filaments? In this study, we use high-speed AFM to evaluate this issue. We observed that the initial binding of small arrays of myosin heads to the non-regulated actin filaments did not affect the cooperative probability of subsequent bindings and did not lead to an increase in the fractional occupancy of the actin binding sites. These results suggest that myosin motors are independent force generators when connected in small arrays, and that the binding of one myosin does not alter the kinetics of other myosins. In contrast, the probability of binding of myosin heads to regulated thin filaments under activating conditions (at high Ca2+ concentration in the presence of 2 μM ATP) was increased with the initial binding of one myosin, leading to a larger occupancy of available binding sites at the next half-helical pitch of the filament. The result suggests that myosin cooperativity is observed over five pseudo-repeats and defined by the activation status of the thin filaments.

Relationship of binding-site occupancy, transthyretin stabilisation and disease modification in patients with tafamidis-treated transthyretin amyloid cardiomyopathy

Background Tafamidis inhibits progression of transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) by binding TTR tetramer and inhibiting dissociation to monomers capable of denaturation and deposition in cardiac tissue. While the phase 3 ATTR-ACT trial demonstrated the efficacy of tafamidis, the degree to which the approved dose captures the full potential of the mechanism has yet to be assessed. Methods We developed a model of dynamic TTR concentrations in plasma to relate TTR occupancy by tafamidis to TTR stabilisation. We then developed population pharmacokinetic–pharmacodynamic models to characterise the relationship between stabilisation and measures of disease progression. Results Modelling individual patient data of tafamidis exposure and increased plasma TTR confirmed that single-site binding provides complete tetramer stabilisation in vivo. The approved dose was estimated to reduce unbound TTR tetramer by 92%, and was associated with 53%, 56% and 49% decreases in the rate of change in NT-proBNP, KCCQ-OS, and six-minute walk test disease progression measures, respectively. Simulating complete TTR stabilisation predicted slightly greater reductions of 58%, 61% and 54%, respectively. Conclusions These findings support the value of TTR stabilisation as a clinically beneficial treatment option in ATTR-CM and the ability of tafamidis to realise nearly the full therapeutic benefit of this mechanism. Clinicaltrials.gov identifier NCT01994889

Relationship of tafamidis binding site occupancy, transthyretin stabilization, and disease modification in tafamidis treated transthyretin amyloid cardiomyopathy patients

Abstract Introduction Tafamidis inhibits progression of transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) by binding TTR tetramer and inhibiting its dissociation to monomers which can denature and result in amyloid fibril formation and deposition in cardiac tissue. Purpose While the phase 3 ATTR-ACT clinical data clearly demonstrated efficacy, quantification of TTR stabilization in patients and the degree to which the approved dose captures the full potential of the mechanism has yet to be assessed. Methods Isothermal titration calorimetry and subunit fraction exchange were used to determine affinities to the two binding sites on TTR. These values were used to develop a model of tafamidis binding to TTR in plasma that was applied to individual patient data to calculate TTR binding site occupancy and the change in total TTR levels induced by TTR stabilization. Population pharmacodynamic (PD) models were developed for three measures of disease progression, plasma NT-proBNP levels, Kansas City Cardiomyopathy Questionnaire – Overall Score (KCCQ-OS), and six-minute walk test (6MWT) distance, to evaluate patient response with the degree of TTR occupancy. Results In vitro binding data of wild-type TTR confirmed tafamidis binds two sites of TTR with negative cooperativity and provided precise estimates of the binding affinity to TTR and albumin. Modeling individual patient data of tafamidis exposure and increased TTR plasma levels using the in vitro derived binding affinity values confirms single site binding is consistent with complete tetramer stabilization. Patients given 80 mg tafamidis meglumine, the clinically approved dose for ATTR-CM, had a 92% reduction in unbound, unstabilized TTR, which correlated with a 53% decrease in NT-proBNP elevation, a 56% decrease in KCCQ-OS worsening and a 49% reduced decline in the 6MWT. For 100% receptor occupancy and stabilization, the expected effects on these measures are 58%, 61%, and 54% for NT-proBNP, KCCQ-OS, and 6MWT, respectively. Conclusions These results demonstrate a quantitative relationship between TTR stabilization, the mechanism of action of tafamidis, and accepted laboratory and patient-based outcomes in ATTR-CM. These results also support the value of TTR stabilization as a clinically beneficial treatment option which maintains the protein in its physiologically active form within the body. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): This study was sponsored by Pfizer.

Structural and dynamic mechanisms of GABAA receptor modulators with opposing activities

γ-Aminobutyric acid type A (GABAA) receptors are pentameric ligand-gated ion channels abundant in the central nervous system and are prolific drug targets for treating anxiety, sleep disorders and epilepsy. Diverse small molecules exert a spectrum of effects on γ-aminobutyric acid type A (GABAA) receptors by acting at the classical benzodiazepine site. They can potentiate the response to GABA, attenuate channel activity, or counteract modulation by other ligands. Structural mechanisms underlying the actions of these drugs are not fully understood. Here we present two high-resolution structures of GABAA receptors in complex with zolpidem, a positive allosteric modulator and heavily prescribed hypnotic, and DMCM, a negative allosteric modulator with convulsant and anxiogenic properties. These two drugs share the extracellular benzodiazepine site at the α/γ subunit interface and two transmembrane sites at β/α interfaces. Structural analyses reveal a basis for the subtype selectivity of zolpidem that underlies its clinical success. Molecular dynamics simulations provide insight into how DMCM switches from a negative to a positive modulator as a function of binding site occupancy. Together, these findings expand our understanding of how GABAA receptor allosteric modulators acting through a common site can have diverging activities.

High-Efficiency CdSe Quantum Dots/Fe3O4@MoS2/S2O82- Electrochemiluminescence System Based on a Microfluidic Analysis Platform for the Sensitive Detection of Neuron-Specific Enolase.

In this work, based on electrochemiluminescence (ECL) technology and self-assembled portable disease detection chips, a bioactivity-maintained sensing platform was developed for the quantitative detection of neuron-specific enolase. First, we prepared Fe3O4@MoS2 nanocomposites as an efficient catalyst to accelerate the reduction of persulfate (S2O82-). Specifically, abundant sulfate radicals (SO4•-) were generated because of cyclic conversion between Fe2+ and Fe3+. Meanwhile, MoS2 nanoflowers with a high specific surface area could not only load more Fe3O4 but also solve its agglomeration problem, which greatly improved the catalytic efficiency. Moreover, a biosensor chip was constructed by standard lithography processes for disease detection, which had good sensitivity and portability. According to the above strategies, the developed portable sensing platform played the part of promoting the practical application of bioanalysis in early tumor screening and clinical diagnosis. In addition, we developed a short peptide ligand (NARKFYKG, NAR) to avoid the occupation of antigen binding sites by specifically connecting to Fc fragments in antibodies. Thus, the binding efficiency of antibodies and the activity of biosensors were improved due to the introduction of NAR.

Probing function in ligand-gated ion channels without measuring ion transport.

Although the functional properties of ion channels are most accurately assessed using electrophysiological approaches, a number of experimental situations call for alternative methods. Here, working on members of the pentameric ligand-gated ion channel (pLGIC) superfamily, we focused on the practical implementation of, and the interpretation of results from, equilibrium-type ligand-binding assays. Ligand-binding studies of pLGICs are by no means new, but the lack of uniformity in published protocols, large disparities between the results obtained for a given parameter by different groups, and a general disregard for constraints placed on the experimental observations by simple theoretical considerations suggested that a thorough analysis of this classic technique was in order. To this end, we present a detailed practical and theoretical study of this type of assay using radiolabeled α-bungarotoxin, unlabeled small-molecule cholinergic ligands, the human homomeric α7-AChR, and extensive calculations in the framework of a realistic five-binding-site reaction scheme. Furthermore, we show examples of the practical application of this method to tackle two longstanding questions in the field: our results suggest that ligand-binding affinities are insensitive to binding-site occupancy and that mutations to amino-acid residues in the transmembrane domain are unlikely to affect the channel's affinities for ligands that bind to the extracellular domain.

Transcription activation is enhanced by multivalent interactions independent of phase separation

Transcription factors (TFs) consist of a DNA-binding domain and an activation domain (AD) that are frequently considered to be independent and exchangeable modules. However, recent studies report that the physicochemical properties of the AD can control TF assembly at chromatin by driving phase separation into transcriptional condensates. Here, we dissected transcription activation by comparing different synthetic TFs at a reporter gene array with real-time single-cell fluorescence microscopy. In these experiments, binding site occupancy, residence time, and coactivator recruitment in relation to multivalent TF interactions were compared. While phase separation propensity and activation strength of the AD were linked, the actual formation of liquid-like TF droplets had a neutral or inhibitory effect on transcription activation. We conclude that multivalent AD-mediated interactions enhance the transcription activation capacity of a TF by increasing its residence time in the chromatin-bound state and facilitating the recruitment of coactivators independent of phase separation.

Unveiling the Mechanisms of High-Temperature 1/2[111] Screw Dislocation Glide in Iron–Carbon Alloys

We have developed a self-consistent model for predicting the velocity of 1/2[111] screw dislocation in binary iron–carbon alloys gliding by a high-temperature Peierls mechanism. The methodology of modelling includes: (i) Kinetic Monte-Carlo (kMC) simulation of carbon segregation in the dislocation core and determination the total carbon occupancy of the core binding sites; (ii) Determination of kink-pair formation enthalpy of a screw dislocation in iron—carbon alloy; (iii) KMC simulation of carbon drag and determination of maximal dislocation velocity at which the atmosphere of carbon atoms can follow a moving screw dislocation; (iv) Self consistent calculation of the average velocity of screw dislocation in binary iron–carbon alloys gliding by a high-temperature kink-pair mechanism under a constant strain rate. We conduct a quantitative analysis of the conditions of stress and temperature at which screw dislocation glide in iron–carbon alloy is accomplished by a high-temperature kink-pair mechanism. We estimate the dislocation velocity at which the screw dislocation breaks away from the carbon cloud and thermally-activated smooth dislocation propagation is interrupted by sporadic bursts of dislocation activity.

In Silico Approaches for the Identification of Novel Inhibitors against Breast Cancer Up-Regulated Protein

Breast cancer is a type of cancer that develops in the breast tissues. When some breast cells begin to grow abnormally, breast cancer develops. These cells grow and divide at a faster rate than healthy cells and continue to grow, generating a lump or mass. Cancer cells in the breast may spread to lymph nodes or other places of the body. The hormone estrogen encourages cancer growth when it binds to the receptor of the targeted protein. The purpose of this study is the rational screening of a 15,000 phytochemicals library against the estrogen receptor alpha protein. The library was employed for molecular docking to find the binding affinities and simulation analysis of the top-selected compounds. The top four compounds, Mangostenone E, Exiguaflavanone M, Sanggenon A, and Flaccidine were identified as direct inhibitors of estrogen receptors as evident from their high binding affinity and occupancy of specific binding sites. Mangostenone E was the leading phytochemical that showed a high docking score—15.97 (kcal/mol)—and bonding interaction at the active site of Mangostenone E. Leading phytochemicals were subjected to analysis for drug-like properties that further reinforced their validation. Potential molecules identified in this study can be considered lead drugs for the treatment of breast cancer. KEYWORDS Bioinformatics, docking, drug candidates, molecular dynamic simulation, phytochemicals, protein data bank

Weak multivalent biomolecular interactions: a strength versus numbers tug of war with implications for phase partitioning.

In this short Perspective, we discuss how recent dynamic live-cell imaging experiments have challenged our understanding of mechanisms driving functional molecular interactions in vivo. While we have generally considered the formation of functional biomolecular complexes as resulting from the stable assembly of two or more partner molecules, here we entertain the possibility that function may actually be maintained while molecules are rapidly exchanged within a complex. We postulate that at high effective concentrations, even very weak interactions can lead to strong binding site occupancy and thereby mediate function in a highly dynamic fashion. This new perspective in our definition of what represents a functional complex in living cells and in vivo could significantly alter how we define the nature of molecular transactions critical for mediating regulation in the cellular context. These less conventional principles also allow a broadening of the mechanistic options we should explore when interpreting essential biological processes such as gene regulation.

Measuring Affinity of Ligands to the Oxytocin Receptor Using Radioligand Binding.

Two aims of oxytocin receptor (OTR)-targeted drug discovery are development of selective OTR-binding PET tracers and development of brain-permeable selective OTR agonists. By allowing measurement of central OTR binding site occupancy after administration of intranasal oxytocin, OTR PET tracers inform anunderstanding of the conflicting effects on pro-social behaviors seenwith administration of intranasal oxytocin in human studies. By mitigating pharmacokinetic and pharmacodynamic limitations of intranasal oxytocin, development of brain-permeable selective OTR agonists may produce therapies for mental disorders that involve asocial symptoms. A key step in development of new OTR-targeting PET radioligands and small molecule agonists is measurement of OTR affinity. One technique that can quantitate the affinity of candidate ligands for the OTR is competition radioligand binding. This chapter describes the materials, methods, and considerations of experimental design required to conduct the steps of competition radioligand binding for OTR drug discovery.