Change In Binding Affinity Research Articles

Evaluation and Characterization of Trk Kinase Inhibitors for the Treatment of Pain: Reliable Binding Affinity Predictions from Theory and Computation.

Optimization of ligand binding affinity to the target protein of interest is a primary objective in small-molecule drug discovery. Until now, the prediction of binding affinities by computational methods has not been widely applied in the drug discovery process, mainly because of its lack of accuracy and reproducibility as well as the long turnaround times required to obtain results. Herein we report on a collaborative study that compares tropomyosin receptor kinase A (TrkA) binding affinity predictions using two recently formulated fast computational approaches, namely, Enhanced Sampling of Molecular dynamics with Approximation of Continuum Solvent (ESMACS) and Thermodynamic Integration with Enhanced Sampling (TIES), to experimentally derived TrkA binding affinities for a set of Pfizer pan-Trk compounds. ESMACS gives precise and reproducible results and is applicable to highly diverse sets of compounds. It also provides detailed chemical insight into the nature of ligand-protein binding. TIES can predict and thus optimize more subtle changes in binding affinities between compounds of similar structure. Individual binding affinities were calculated in a few hours, exhibiting good correlations with the experimental data of 0.79 and 0.88 from the ESMACS and TIES approaches, respectively. The speed, level of accuracy, and precision of the calculations are such that the affinity predictions can be used to rapidly explain the effects of compound modifications on TrkA binding affinity. The methods could therefore be used as tools to guide lead optimization efforts across multiple prospective structurally enabled programs in the drug discovery setting for a wide range of compounds and targets.

AFIR: A Dimensionless Potency Metric for Characterizing the Activity of Monoclonal Antibodies.

For monoclonal antibody (mAb) drugs, soluble targets may accumulate several thousand fold after binding to the drug. Time course data of mAb and total target is often collected and, although free target is more closely related to clinical effect, it is difficult to measure. Therefore, mathematical models of this data are used to predict target engagement. In this article, a “potency factor” is introduced as an approximation for the model‐predicted target inhibition. This potency factor is defined to be the time‐Averaged Free target concentration to Initial target concentration Ratio (AFIR), and it depends on three key quantities: the average drug concentration at steady state; the binding affinity; and the degree of target accumulation. AFIR provides the intuition for how changes in dosing regimen and binding affinity affect target capture and AFIR can be used to predict the druggability of new targets and the expected benefits of more potent, second‐generation mAbs.

Effect of Transient Helicity of cMyb TAD on its Binding Affinity to the Kix Domain of CBP/p300

Intrinsically disordered proteins (IDPs) are involved in various protein‐protein interactions and they often experience a coupled folding and binding reaction when they bind to their protein partners. Many IDPs, like the cMyb trans activation domain (cMyb TAD), exhibit transient helical structures in solution that resemble their bound structures. We hypothesize that the levels of transient helicity in the free IDP will affect the binding affinity to their protein partners in a predictable way. Previous studies have shown that increasing the transient helicity of intrinsically disordered p53 tumor suppressor increases the binding affinity to MDM2 and had negative effects on cellular function. We aim to study the effect of changing cMyb TAD transient helicity on its binding affinity to the kix domain of CBP/p300.cMyb is a hematopoietic regulator which is known to play a role in tumorigenesis. Unlike p53 TAD, cMyb TAD has a high residual helicity in solution in the absence of binding partners. Prolines that flank the N and C‐terminus of this helical region were mutated to alanines to determine whether there was a change in helicity that would affect the binding of Kix. Upon mutation, the increase in helicity had almost no effect on the binding affinity of the cMyb‐Kix complex. Further, mutations that reduce the helicity of cMyb‐TAD were designed to test the effect of reducing transient helicity on the binding affinity to Kix. To achieve this goal, conservative mutations of the solvent exposed amino acids in cMyb were made. We are currently measuring the transient helicity and binding affinity of these mutants with the long‐range goal of identifying a general relationship between changes in transient helicity and binding affinity of IDPs.Support or Funding InformationThis research was supported by funding to GWD by National Institutes of Health (2R01CA14124406‐A1 and 1R01GM115556‐01A1). This work was also supported by funding to JC by Wellcome trust (WT 095195MA) and by a Biotechnology and Biological Sciences Research Council (BBSRC) studentship to MDC.

Residues W320 and Y328 within the binding site of the μ-opioid receptor influence opiate ligand bias

The development of G protein-biased agonists for the μ-opioid receptor (MOR) offers a clear drug discovery rationale for improved analgesia and reduced side-effects of opiate pharmacotherapy. However, our understanding of the molecular mechanisms governing ligand bias is limited, which hinders our ability to rationally design biased compounds. We have investigated the role of MOR binding site residues W320 and Y328 in controlling bias, by receptor mutagenesis. The pharmacology of a panel of ligands in a cAMP and a β-arrestin2 assay were compared between the wildtype and mutated receptors, with bias factors calculated by operational analysis using ΔΔlog(τ/KA) values. [3H]diprenorphine competition binding was used to estimate affinity changes. Introducing the mutations W320A and Y328F caused changes in pathway bias, with different patterns of change between ligands. For example, DAMGO increased relative β-arrestin2 activity at the W320A mutant, whilst its β-arrestin2 response was completely lost at Y328F. In contrast, endomorphin-1 gained activity with Y328F but lost activity at W320A, in both pathways. For endomorphin-2 there was a directional shift from cAMP bias at the wildtype towards more β-arrestin2 bias at W320A. We also observe clear uncoupling between mutation-driven changes in function and binding affinity. These findings suggest that the mutations influenced the balance of pathway activation in a ligand-specific manner, thus identifying residues in the MOR binding pocket that govern ligand bias. This increases our understanding of how ligand/receptor binding interactions can be translated into agonist-specific pathway activation.

CaMKII Control of Actin Cytoskeletal Dynamics

The calcium calmodulin dependent kinase (CaMKII) binds and organizes actin Binding is abolished by calcium calmodulin, triggering cytoskeletal remodeling with important implications for synaptic transmission in dendritic spines; CaMKII and actin-rich post-synaptic micro-compartments that expand within one minute upon synaptic stimulation to initiate long-term changes associated with learning. CaMKII organizes static F-actin bundles in-vitro, but fluorescence microscopy single particle tracking (SPT) of GFP-tagged CaMKII holoenzymes (GFP-CaMKII) in spines has not detected stimulus-dependent jumps in mobility consistent with un-bundling. However, changes in binding affinities, in response to osmotic and mechanical forces, might provoke diverse cytoskeletal CaMKII-actin architectures in vivo. To look for these, we initially used SPT in live cells and found both rat neuronal CaMKII isoforms bound RFP-actin labeled stress fibers weakly, comparable to G-actin, with strong binding to the fibers obtained only when multiple holoenzyme subunits are engaged (Khan et al. 2016. Biophys. J.111,395-408). We have now measured the binding of GFP-CaMKII molecules to rhodamine-phalloidin labeled, single F-actin filaments and characterized the architectural transitions of the formed filament networks to mechano-osmotic stress. CaMKII induced formation of mechanically-resilient networks with aster-like nodes. Cryo-electron tomography showed CaMKII localized in small clusters (n < 5) at the nodes. Although nodes disappeared rapidly upon addition of calcium calmodulin, the network structure took several minutes to dissipate. Both C. elegans and human CaMKIIs formed networks at stoichiometric concentrations, revealing an evolutionary-conserved interaction. At the micromolar concentrations investigated, CaMKII-actin bundles were sparsely distributed in the networks. Macromolecular crowding by addition of polyethylene glycol or force generated by addition of myosin motors plus ATP bundled the filaments; but CaMKII retarded, rather than assisted, this transition. CaMKII also protected against filament fragmentation by motor forces. We propose that stimulation-triggered disruption of spine CaMKII-actin interactions has two distinct effects, formation of a compliant F-actin network primarily due to loss of nodal contacts and polymerization of the released G-actin. We are simulating these processes in a 3-dimensional “virtual” spine to determine CaMKII, G and F-actin concentrations and affinities that best mimic the observed spine dynamics upon stimulation.

Molecular Effects of Cardiac Troponin DCM Mutations on Calcium Sensitivity and Myofilament Activation - an Integrated Multi-Scale Modeling Study

Mutations in cTnC (D75Y, E59D and G159D), a key regulatory protein of myofilament contraction, have been associated with dilated cardiomyopathy (DCM). Despite reports of altered myofilament function in these mutants, the underlying molecular alterations caused by these mutations remain elusive. Here we investigate in silico the intra-molecular mechanisms (both structure and dynamics) by which these mutations affect myofilament contraction (i.e. function). Based on the location of cTnC mutations, we tested the hypothesis that intra-molecular effects can explain the altered myofilament calcium sensitivity of force development for D75Y and E59D cTnC, whereas altered cTnC-cTnI interaction contributes to the reported contractile effects of the G159D mutation. We employed a multi-scale approach combining molecular dynamics (MD) and Brownian dynamics (BD) simulations to estimate cTnC calcium association and hydrophobic patch opening. We then integrated these parameters into a Markov model of myofilament activation to compute the steady-state force-pCa relationship. The analysis showed that myofilament calcium sensitivity with D75Y and E59D can be explained by changes in calcium binding affinity of cTnC and the rate of hydrophobic patch opening, if a partial cTnC interhelical opening angle (110°) is sufficient for cTnI switch peptide association to cTnC. In contrast, interactions between cTnC and cTnI within the cardiac troponin complex must also be accounted for to explain contractile alterations due to G159D. In conclusion, this is the first multi-scale in silico study to elucidate how direct molecular effects of genetic mutations in cTnC translate to altered myofilament contractile function.

Robust Ligand Binding to the Protein Translocation Complex (Secyeg) Requires a Lipid Environment

Many secretory proteins pass the membrane barrier via the bacterial SecY complex. SecY is assisted by the motor protein SecA, which is thought to push the polypeptides through the SecY pore with its two-helix finger. This model requires ATP molecules to exchange continuously during translocation. Whether the resulting change in SecA-SecY binding affinity leads to SecA release is unknown. Here we have tested the hypothesis by monitoring SecA binding to the SecY complex using lanthanide resonance energy transfer between a fluorescent label on the tip of its two-helix finger and Tb3+ in a genetically engineered binding pocket on SecY. In the absence of ATP, binding only occurred to SecY molecules that were reconstituted into lipid bilayers, but not to SecY molecules in detergent. In a fraction of these binding events we observed a deep insertion of SecA's two-helix finger into the SecY-channel. These penetrations resulted in relocation of SecY's plug as indicated by measurements of the distance between fingertip and plug. The ‘‘plug’’ is a short helix that is (i) normally located in the center of the channel adjacent to its hydrophobic constriction zone (pore ring) and (ii) blocks the passage of small molecules through the idle SecY complex (1). Observation of ion channel activity in reconstituted planar lipid bilayers confirmed that the plug had moved out of the pore. As previously observed in the case of ribosome nascent chain complexes (2), physiological values of the membrane potential were required to close the channel. We conclude that (i) SecA binding is sufficient to open the protein translocation channel in its lipid environment and (ii) the current transport model needs to be revised to account for SecY's voltage sensitivity.The project was supported by the Austrian Science Fund (P25844, P28213).1. Saparov SM,..,Pohl P (2007) Mol Cell 26:501-509.2. Knyazev DG, ⋯, Pohl P (2014) J Biol Chem 289:24611-24616.

A Quantitative Model to Estimate Drug Resistance in Pathogens.

Pneumocystis pneumonia (PCP) is an opportunistic infection that occurs in humans and other mammals with debilitated immune systems. These infections are caused by fungi in the genus Pneumocystis, which are not susceptible to standard antifungal agents. Despite decades of research and drug development, the primary treatment and prophylaxis for PCP remains a combination of trimethoprim (TMP) and sulfamethoxazole (SMX) that targets two enzymes in folic acid biosynthesis, dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), respectively. There is growing evidence of emerging resistance by Pneumocystis jirovecii (the species that infects humans) to TMP-SMX associated with mutations in the targeted enzymes. In the present study, we report the development of an accurate quantitative model to predict changes in the binding affinity of inhibitors (Ki, IC50) to the mutated proteins. The model is based on evolutionary information and amino acid covariance analysis. Predicted changes in binding affinity upon mutations highly correlate with the experimentally measured data. While trained on Pneumocystis jirovecii DHFR/TMP data, the model shows similar or better performance when evaluated on the resistance data for a different inhibitor of PjDFHR, another drug/target pair (PjDHPS/SMX) and another organism (Staphylococcus aureus DHFR/TMP). Therefore, we anticipate that the developed prediction model will be useful in the evaluation of possible resistance of the newly sequenced variants of the pathogen and can be extended to other drug targets and organisms.

A novel method for in silico identification of regulatory SNPs in human genome

Regulatory single nucleotide polymorphisms (rSNPs), kind of functional noncoding genetic variants, can affect gene expression in a regulatory way, and they are thought to be associated with increased susceptibilities to complex diseases. Here a novel computational approach to identify potential rSNPs is presented. Different from most other rSNPs finding methods which based on hypothesis that SNPs causing large allele-specific changes in transcription factor binding affinities are more likely to play regulatory functions, we use a set of documented experimentally verified rSNPs and nonfunctional background SNPs to train classifiers, so the discriminating features are found. To characterize variants, an extensive range of characteristics, such as sequence context, DNA structure and evolutionary conservation etc. are analyzed. Support vector machine is adopted to build the classifier model together with an ensemble method to deal with unbalanced data. 10-fold cross-validation result shows that our method can achieve accuracy with sensitivity of ~78% and specificity of ~82%. Furthermore, our method performances better than some other algorithms based on aforementioned hypothesis in handling false positives. The original data and the source matlab codes involved are available at https://sourceforge.net/projects/rsnppredict/.

BindProfX: Assessing Mutation-Induced Binding Affinity Change by Protein Interface Profiles with Pseudo-Counts

Understanding how gene-level mutations affect the binding affinity of protein–protein interactions is a key issue of protein engineering. Due to the complexity of the problem, using physical force field to predict the mutation-induced binding free-energy change remains challenging. In this work, we present a renewed approach to calculate the impact of gene mutations on the binding affinity through the structure-based profiling of protein–protein interfaces, where the binding free-energy change (ΔΔG) is counted as the logarithm of relative probability of mutant amino acids over wild-type ones in the interface alignment matrix; three pseudo-counts are introduced to alleviate the limit of the current interface library. Compared with a previous profile score that was based on the log-odds likelihood calculation, the correlation between predicted and experimental ΔΔG of single-site mutations is increased in this approach from 0.33 to 0.68. The structure-based profile score is found complementary to the physical potentials, where a linear combination of the profile score with the FoldX potential could increase the ΔΔG correlation from 0.46 to 0.74. It is also shown that the profile score is robust for counting the coupling effect of multiple individual mutations. For the mutations involving more than two mutation sites where the correlation between FoldX and experimental data vanishes, the profile-based calculation retains a strong correlation with the experimental measurements.

Guide Strand 3'-End Modifications Regulate siRNA Specificity.

Short interfering RNA (siRNA)-triggered gene knockdown through the RNA interference (RNAi) pathway is widely used to study gene function, and siRNA-based therapeutics are in development. However, as the guide strand of an siRNA can function like a natural microRNA (miRNA), siRNAs often repress hundreds of off-target transcripts with complementarity only to the seed region (nucleotides 2-8) of the guide strand. Here, we describe novel guide strand 3'-end modifications derived from 1-ethynylribose (1-ER) and copper-catalyzed azide-alkyne cycloaddition reactions and evaluate their impact on target versus miRNA-like off-target knockdown. Surprisingly, when positioned at the guide strand 3'-end, the parent 1-ER modification substantially reduced off-target knockdown while having no measurable effect on on-target knockdown potency. In addition, these modifications were shown to modulate siRNA affinity for the hAgo2 PAZ domain. However, the change in PAZ domain binding affinity was not sufficient to predict the modification's effect on miRNA-like off targeting.

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa.

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.

On the fly estimation of host-guest binding free energies using the movable type method: participation in the SAMPL5 blind challenge.

We review our performance in the SAMPL5 challenge for predicting host-guest binding affinities using the movable type (MT) method. The challenge included three hosts, acyclic Cucurbit[2]uril and two octa-acids with and without methylation at the entrance to their binding cavities. Each host was associated with 6-10 guest molecules. The MT method extrapolates local energy landscapes around particular molecular states and estimates the free energy by Monte Carlo integration over these landscapes. Two blind submissions pairing MT with variants of the KECSA potential function yielded mean unsigned errors of 1.26 and 1.53kcal/mol for the non-methylated octa-acid, 2.83 and 3.06kcal/mol for the methylated octa-acid, and 2.77 and 3.36kcal/mol for Cucurbit[2]uril host. While our results are in reasonable agreement with experiment, we focused on particular cases in which our estimates gave incorrect results, particularly with regard to association between the octa-acids and an adamantane derivative. Working on the hypothesis that differential solvation effects play a role in effecting computed binding affinities for the parent octa-acid and the methylated octa-acid and that the ligands bind inside the pockets (rather than on the surface) we devised a new solvent accessible surface area term to better quantify solvation energy contributions in MT based studies. To further explore this issue a, molecular dynamics potential of mean force (PMF) study indicates that, as found by our docking calculations, the stable binding mode for this ligand is inside (rather than surface bound) the octa-acid cavity whether the entrance is methylated or not. The PMF studies also obtained the correct order for the methylation-induced change in binding affinities and associated the difference, to a large extent to differential solvation effects. Overall, the SAMPL5 challenge yielded in improvements our solvation modeling and also demonstrated the need for thorough validation of input data integrity prior to any computational analysis.

Tuning the Binding Affinities and Reversion Kinetics of a Light Inducible Dimer Allows Control of Transmembrane Protein Localization.

Inducible dimers are powerful tools for controlling biological processes through colocalizing signaling molecules. To be effective, an inducible system should have a dissociation constant in the "off" state that is greater (i.e., weaker affinity) than the concentrations of the molecules that are being controlled, and in the "on" state a dissociation constant that is less (i.e., stronger affinity) than the relevant protein concentrations. Here, we reengineer the interaction between the light inducible dimer, iLID, and its binding partner SspB, to better control proteins present at high effective concentrations (5-100 μM). iLID contains a light-oxygen-voltage (LOV) domain that undergoes a conformational change upon activation with blue light and exposes a peptide motif, ssrA, that binds to SspB. The new variant of the dimer system contains a single SspB point mutation (A58V), and displays a 42-fold change in binding affinity when activated with blue light (from 3 ± 2 μM to 125 ± 40 μM) and allows for light-activated colocalization of transmembrane proteins in neurons, where a higher affinity switch (0.8-47 μM) was less effective because more colocalization was seen in the dark. Additionally, with a point mutation in the LOV domain (N414L), we lengthened the reversion half-life of iLID. This expanded suite of light induced dimers increases the variety of cellular pathways that can be targeted with light.

Predicted structure of a Minus-C OBP from Batocera horsfieldi (Hope) suggests an intermediate structure in evolution of OBPs.

Odorant binding proteins (OBPs) transport hydrophobic odorants from the environment to odorant receptors and play an important role in specific recognition of volatiles. Here, we expressed and purified a minus-C OBP, BhorOBPm2, from Batocera horsfieldi, a major pest of Popolus, to determine its binding characteristics with 58 candidate volatiles using a fluorescence competition-binding assay. We showed that BhorOBPm2 exhibited high binding affinity with chain volatiles and that ligands were selected based on chain length. In order to elucidate the binding mechanism, homology modeling and molecular-docking experiments were performed to investigate interactions between BhorOBPm2 and volatiles. The predicted structure with only two disulfide bonds showed one continuous channel for ligand binding, similar to classic OBPs AgamOBP1 and CquiOBP1. Unexpectedly, we observed a larger binding pocket for BhorOBPm2 and broader specificity for ligands than classic OBPs due to the expansive flexibility of BhorOBPm2 resulting from a lack of disulfide bonds. These findings suggested that BhorOBPm2 might present an intermediate structure in the evolution of OBPs. Furthermore, we designed two mutant proteins to simulate and verify functions of the C-terminal region. The changes in binding affinity observed here indicated a novel action differing from that of the “lid” described in previous studies.

Unravelling the binding mechanism and protein stability of human serum albumin while interacting with nefopam analogues: a biophysical and insilico approach

In this study, molecular binding affinity was investigated for Nefopam analogues (NFs), a functionalized benzoxazocine, with human serum albumin (HSA), a major transport protein in the blood. Its binding affinity and concomitant changes in its conformation, binding site and simulations were also studied. Fluorescence data revealed that the fluorescence quenching of HSA upon binding of NFs analogues is based on a static mechanism. The three analogues of NFs binding constants (KA) are in the order of NF3 > NF2 > NF1 with values of 1.53 ± .057 × 104, 2.16 ± .071 × 104 and 3.6 ± .102 × 105 M−1, respectively. Concurrently, thermodynamic parameters indicate that the binding process was spontaneous, and the complexes were stabilized mostly by hydrophobic interactions, except for NF2 has one hydrogen bond stabilizes it along with hydrophobic interactions. Circular dichroism (CD) studies revealed that there is a decrease in α-helix with an increase in β-sheets and random coils signifying partial unfolding of the protein upon binding of NFs, which might be due to the formation of NFs-HSA complexes. Further, molecular docking studies showed that NF1, NF2 and NF3 bound to subdomains IIIA, IB and IIA through hydrophobic interactions. However, NF1 have additionally formed a single hydrogen bond with LYS 413. Furthermore, molecular simulations unveiled that NFs binding was in support with the structural perturbation observed in CD, which is evident from the root mean square deviation and Rg fluctuations. We hope our insights will provide ample scope for engineering new drugs based on the resemblances with NFs for enhanced efficacy with HSA.

Protein kinase C-δ inhibitor, Rottlerin inhibits growth and survival of mycobacteria exclusively through Shikimate kinase

The molecular bases of disease provide exceptional prospect to translate research findings into new drugs. Nevertheless, to develop new and novel chemical entities takes huge amount of time and efforts, mainly due to the stringent processes. Therefore, drug repurposing is one of such strategies which is being used in recent times to identify new pharmacophores. The essential first step in discovery of the specific inhibitor with low toxicity is the identification and elucidation of pathways exclusive to target pathogen. One such target is the shikimate pathway, which is essential for algae, higher plants, bacteria and fungi. Since, this enzyme system is absent in higher eukaryotes and in mammals, the enzymes involved in the pathway provide an attractive target for the development of potentially selective and non toxic antimicrobial agents. Since, so far there is no specific inhibitor which is able to restrain mycobacterial shikimate pathway; we expanded the use of a known kinase inhibitor; Rottlerin, in order to predict the prototype in discovering the specific molecules against this enzyme. For the first time we have shown that Rottlerin inhibits extracellular mycobacteria by affecting Shikimate Kinase (SK) and this effect is further enhanced during the intracellular infection due to the added effect of PKC- δ down-regulation. The molecular docking of Rottlerin with both the mycobacterial SKs, corroborated the inhibition data, and revealed that the effects of SK, in slow and in fast grower mycobacteria are due to the changes in affinity of binding with the drug.

Synthesis and Evaluation of a New Series of 8-(2-Nitroaryl)Xanthines as Adenosine Receptor Ligands.

Preclinical Research A new series of 1,3-dimethylxanthine derivatives bearing 8-(2-nitroaryl) residue was synthesized and evaluated for affinity for recombinant human adenosine receptors subtypes. Nitrate esters of 7-substituted-1,3-dimethyl-8-phenylxanthines were also synthesized and tested. Introducing a nitro substituent at the 2-position of the 8-substituted phenyl ring resulted in generally low affinity for adenosine receptors (ARs), selectivity toward the A2A subtype was enhanced in some of the compounds. 8-(4-Cyclopentyloxy-5-methoxy-2-nitrophenyl)-1,3-dimethylxanthine (9e) proved to be a potent compound among the 2-nitrophenyl substituted xanthines exhibiting a Ki = 1 μM at human A2A ARs with at least 30 fold selectivity versus human A1 and A2B ARs. Replacement of 8-chloropropoxy phenyl with 8-nitrooxypropoxy phenyl resulted in a negligible change in binding affinity of the 8-substituted xanthines for various AR subtypes. Drug Dev Res 77 : 241-250, 2016. © 2016 Wiley Periodicals, Inc.

Cortisol and CBG - Getting cortisol to the right place at the right time.

Cortisol is transported in the blood by corticosteroid-binding globulin (CBG), a non-inhibitory member of the serpin family of serine protease inhibitors. Recent structural advances reveal how CBG acts as a releasing-agent as well as a carrier of cortisol. Taken together, the structures of the various forms of CBG and of the closely related thyroxine binding-globulin, show how the inherent conformational mechanism of the serpins has been adapted to modulate hormone release to the tissues by changes in binding affinities. A deduction from this, of the temperature dependence of hormone binding, is remarkably borne out with CBG, with a doubling in plasma free cortisol as the body temperature rises to 39°C. Another insight, against a dogma in the corticosteroid field, is that the proteolytic cleavage of CBG in inflammation results in a partial and not a complete loss of cortisol binding. This becomes of medical importance in conjunction with recent evidence of a pool of the circulating cleaved-form of CBG. It is now evident that tissue levels of free cortisol are buffered by two responsive plasma pools, intact CBG with a high binding-affinity and, particularly in inflammation and sepsis, a further pool of cleaved-CBG with a ten-fold lower affinity. The new molecular understandings, as well as providing insights into the differential release of circulating hormones, also open prospects for therapeutic interventions and draw attention to the potential of CBG and TBG as vehicles for the targeted delivery of drugs.

Exploring the interplay between experimental methods and the performance of predictors of binding affinity change upon mutations in protein complexes.

Reliable prediction of binding affinity changes (ΔΔG) upon mutations in protein complexes relies not only on the performance of computational methods but also on the availability and quality of experimental data. Binding affinity changes can be measured by various experimental methods with different accuracies and limitations. To understand the impact of these on the prediction of binding affinity change, we present the Database of binding Affinity Change Upon Mutation (DACUM), a database of 1872 binding affinity changes upon single-point mutations, a subset of the SKEMPI database (Moal,I.H. and Fernández-Recio,J. Bioinformatics, 2012;28:2600-2607) extended with information on the experimental methods used for ΔΔG measurements. The ΔΔG data were classified into different data sets based on the experimental method used and the position of the mutation (interface and non-interface). We tested the prediction performance of the original HADDOCK score, a newly trained version of it and mutation Cutoff Scanning Matrix (Pires,D.E.V., Ascher,D.B. and Blundell,T.L. Bioinformatics 2014;30:335-342), one of the best reported ΔΔG predictors so far, on these various data sets. Our results demonstrate a strong impact of the experimental methods on the performance of binding affinity change predictors for protein complexes. This underscores the importance of properly considering and carefully choosing experimental methods in the development of novel binding affinity change predictors. The DACUM database is available online at https://github.com/haddocking/DACUM.