Cheng-Prusoff Equation Research Articles

Natural flavonoids as promising lactate dehydrogenase A inhibitors: Comprehensive in vitro and in silico analysis.

The inhibitory potential of 17 flavonoids on lactate dehydrogenase A (LDHA), a key enzyme in the downstream process of aerobic glycolysis in cancer cells, is investigated. Fisetin exhibited excellent inhibitory activity (IC50 = 0.066 µM). Quercetin 3-β-D-glucoside, quercetin 3-galactoside, luteolin, neoeriocitrin, and luteolin 7-O-β-D-glucoside showed good inhibitory activity (IC50 = 1.397-15.730 µM). Biochanin A, baicalein, quercetin, scutellarein-7-glucuronide, diosmetin, baicalein 7-O-β-D-glucuronide, and apigenin 7-apioglucoside demonstrated moderate inhibitory activity (IC50 = 33.007-86.643 µM). Eriodictyol, quercetin 7-O-β-D-glucoside, apigenin 7-O-β-D-glucoside, and epicatechin were inactive. The Lineweaver-Burk plot showed that fisetin competitively inhibits NADH binding (Ki = 0.024 µM). Ki values for other compounds were calculated using the Cheng-Prusoff equation (Ki = 0.2799-2.1661 µM). The study revealed that the inhibitory effect of flavonoids varies with the number and position of OH groups and bound sugars. Molecular docking analyses indicated that flavonoids exhibited strong interactions with the NADH binding site of LDHA through hydrophobic interactions and hydrogen bonds. Molecular dynamic simulations tested the stability of the fisetin-LDHA complex over 100 ns and showed fisetin's high binding affinity to LDHA, maintaining strong hydrogen bonds. The binding energy of fisetin with LDHA was -33.928 kcal/mol, indicating its effectiveness as an LDHA inhibitor. Consequently, flavonoids identified as strong inhibitors could be potential cancer treatment sources through LDHA inhibition.

Binding Curve Viewer: Visualizing the Equilibrium and Kinetics of Protein-Ligand Binding and Competitive Binding.

Understanding the thermodynamics and kinetics of the protein-ligand interaction is essential for biologists and pharmacologists. To visualize the equilibrium and kinetics of the binding reaction with 1:1 stoichiometry and no cooperativity, we obtained the exact relationship of the concentration of the protein-ligand complex and the time in the second-order binding process and numerically simulated the process of competitive binding. First, two common concerns in measuring protein-ligand interactions were focused on how to avoid the titration regime and how to establish the appropriate incubation time. Then, we gave examples of how the commonly used experimental conditions of [L]0 ≫ [P]0 and [I]0 ≫ [P]0 affected the estimation of the kinetic and thermodynamic properties. Theoretical inhibition curves were calculated, and the apparent IC50 and IC50 were estimated accordingly under predefined conditions. Using the estimated apparent IC50, we compared the apparent Ki and Ki calculated by using the Cheng-Prusoff equation, Lin-Riggs equation, and Wang's group equation. We also applied our tools to simulate high-throughput screening and compare the results of real experiments. The visualization tool for simulating the saturation experiment, kinetic experiments of binding and competitive binding, and inhibition curve, "Binding Curve Viewer," is available at www.eplatton.net/binding-curve-viewer.

Natural flavonoids as promising 6-phosphogluconate dehydrogenase inhibitor candidates: In silico and in vitro assessments.

The primary strategy in the fight against cancer is to screen compounds that may be effective on different types of cancer. Compounds from plants seem to be a good source. The present study investigated the inhibitory effects of some flavonoids on the 6-phosphogluconate dehydrogenase (6-PGD) enzyme. We determined that quercetin, myricetin, fisetin, morin, apigenin, and baicalein exhibited powerful inhibition effects with IC50 values between 4.08 and 21.26 µM, while luteolin, kaempferol, apiin, galangin, and baicalin showed moderate effects with IC50 values between 54.15 and 138.91 µM. Quercetin competitively inhibited the binding of NADP and 6-phosphogluconate to the 6-PGD enzyme with Ki values of 0.527 ± 0.251 and 0.374 ± 0.138 µM, respectively. We calculated Ki values using the Cheng-Prusoff equation as between 0.44 and 14.88 µM. The possible interaction details of polyphenols with the active site of 6-PGD were analyzed with docking software. In silico and in vitro studies indicated that the -OH groups on the A and C ring of flavonoids bind to the enzyme's active site via hydrogen bonding, while the -OH groups on the C ring contributed significantly to the increase in the inhibitory potentials of the molecules. Molecular dynamic simulations tested the stability of the 6-PGD-quercetin complex during 100 ns. These phytochemicals were suitable for drug use when optimized with absorption, distribution, metabolism, excretion, and toxicity (ADMET) criteria. The effects of the studied compounds on cancer cell lines of potential targets were demonstrated by network analysis. In conclusion, this study suggests that flavonoids found to be potent inhibitors could serve as leading candidates to treat many cancers via 6-PGD inhibition.

Abstract 2872: Methods of assessment of polysialyltransferase inhibitors for treatment of tumor cell dissemination

Abstract Polysialic acid (polySia) decorates the surface of NCAM (neuronal cell adhesion molecule) on neuroendocrine tumors, notably neuroblastoma and small cell lung cancer, and is strongly associated with poor prognosis and aggressive disease in patients in the clinic [1]. PolySia modulates tumor cell-cell and cell-matrix adhesion, migration, invasion and metastasis. SiRNA knockdown of polysialyltransferase (polyST) ST8SiaII, the enzyme primarily responsible for polySia synthesis in tumors, abrogates tumor cell migration and invasion. PolyST is a selective and largely unexplored therapeutic target for neuroblastoma dissemination [1]. While assays are available to assess polyST enzyme activity, there is no methodology available specifically optimized for identification of novel polyST inhibitors. We describe the development of cell-free and cell-based assays that enable assessment of polysialyltransferase inhibition. Development of the HPLC-fluorescence-based enzyme assay includes a comprehensive optimization of assay conditions, including evaluation of metal ion composition, enzyme concentration, substrate and acceptor concentration, temperature, pH and tolerance to DMSO, followed by validation using known polyST inhibitors. Thorough analysis of each of the assay components provided a set of optimized conditions. Under these optimized conditions, the experimentally observed Ki value for CMP, a competitive polyST inhibitor, was strongly correlated with the predicted Ki value, based on the classical Cheng-Prusoff equation [average fold error (AFE) = 1.043]. These results indicate that this assay can provide medium-throughput analysis for enzyme inhibitors with high accuracy. We additionally report optimized HPLC-based and ELISA-based methodologies for assessment of polyST inhibition in neuroblastoma cells, using endoneuraminidase N as control, and assessment of ICT3176 (a polysialylation inhibitor) as a test agent. In conclusion, in vitro cell-free and cell-based assays for accurate measurement of polysialyltransferase (polyST) inhibition are described, specifically designed for routine identification of potential polyST inhibitors, generation of kinetics data and assessment of mode of inhibition, and assessment of effects on cellular polysialylation. Given the considerable and growing interest in the polySTs as important anti-metastatic targets in cancer drug discovery, these are vital tools to enable preclinical identification and evaluation of novel polyST inhibitors for neuroblastoma therapy. [1] Falconer, R.A. et al., Curr. Cancer Drug Targets, 2012, 12, 925-939. Citation Format: Xiaoxiao Guo, Jodie R. Malcolm, Anjana Patel, Marrwa M. Ali, Goreti Ribeiro Morais, Steven D. Shnyder, Paul M. Loadman, Laurence H. Patterson, Robert A. Falconer. Methods of assessment of polysialyltransferase inhibitors for treatment of tumor cell dissemination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2872.

Discovery of a Lead Brain-Penetrating Gonadotropin-Releasing Hormone Receptor Antagonist with Saturable Binding in Brain.

We report the synthesis, radiosynthesis and biological characterisation of two gonadotropin‐releasing hormone receptor (GnRH−R) antagonists with nanomolar binding affinity. A small library of GnRH−R antagonists was synthesised in 20–67 % overall yield with the aim of identifying a high‐affinity antagonist capable of crossing the blood–brain barrier. Binding affinity to rat GnRH−R was determined by autoradiography in competitive‐binding studies against [125I]buserelin, and inhibition constants were calculated by using the Cheng–Prusoff equation. The radioligands were obtained in 46–79 % radiochemical yield and >95 % purity and with a molar activity of 19–38 MBq/nmol by direct nucleophilic radiofluorination. Positron emission tomography imaging in rat under baseline conditions in comparison to pretreatment with a receptor‐saturating dose of GnRH antagonist revealed saturable uptake (0.1 %ID/mL) into the brain.

Abstract 4186: A biochemical approach to discriminate between ATP-competitive and non-ATP competitive protein kinase inhibitors

Abstract Protein kinases are central to cellular signal transduction and regulation of cellular processes, and are one of the most attractive target classes in modern drug discovery. Multiple kinase inhibitors have already been approved for treatment of various diseases including such severe conditions as cancer. The first and up to today largest group of drugs that effectively inhibits their respective kinase targets belong to the class of ATP competitive compounds. They bind into or near the ATP binding site of the enzymes and inhibit kinase activity by blocking access of ATP to the active site. Although there are numerous examples of highly specific ATP competitive compounds this mode of action is limited by several factors: The ATP binding pocket structures of kinases show a high degree of similarity, which makes finding highly selective compounds challenging. Furthermore, competing with ATP for binding to the same target site, compounds have to be of very high affinity due to the high intracellular ATP concentrations. Therefore, the interest to develop non-ATP competitive inhibitors has risen considerably over the last years. Such inhibitors bind to kinases at sites apart from the ATP binding site, inhibiting their activity e.g. by stabilizing an inactive conformation (like DFG-out state binders), displacing essential cofactors (like cyclins for CDKs) or by blocking activating modifications (like phosphorylation by upstream kinases). We present data of an in-vitro biochemical kinase activity assay setup which is suited to discriminate between ATP-competitive and non-ATP competitive inhibitors. The IC50 of an ATP-competitive inhibitor will increase with increasing ATP concentrations and the IC50 value at a given ATP concentration may be calculated using the equation of Cheng and Prussof: IC50=Ki+(Ki*[ATP]/KM[ATP] (Cheng Y., Prusoff W. H. (1973) Biochem. Pharmacol. 22: 3099-3108). By determining IC50 values for an inhibitor of a specific kinase at different ATP concentrations we examined whether the IC50 value changed according to the Cheng-Prusoff equation, indicating an ATP-competitive mode of action, or if the IC50 values remained unchanged in presence of elevated ATP indicating a non-ATP competitive or mixed type mode of action. In our assay setup we determined the IC50 values at ATP concentrations in a range of 0.1 to 10 fold the ATP KM of the kinase of interest. By comparing the results obtained for the non-ATP competitive MEK1 inhibitor selumetinib and the ATP competitive inhibitor staurosporine we could verify that our assay setup is well suited to discriminate between these different types of kinase inhibitors. Citation Format: Daniel Mueller, Frank Totzke, Thomas Weber, Carolin Heidemann-Dinger, Constance Ketterer, Diane Krämer, Marcel Pathe, Michael H. Kubbutat. A biochemical approach to discriminate between ATP-competitive and non-ATP competitive protein kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4186. doi:10.1158/1538-7445.AM2017-4186

Binding assay for characterization of protein kinase inhibitors possessing sub-picomolar to sub-millimolar affinity

High demand for inhibitors regulating the activity of protein kinases has stimulated the quest for high throughput and reliable compound screening assays. Here we introduce a method applying a non-metal photoluminescent probe ARC-Lum(Fluo) for determination of dissociation constants of competitive inhibitors of protein kinases. Employing a single probe instead of a combination of antibody and fluorescent tracer makes the assay simpler, cheaper, and more accurate than several other inhibitor-screening technologies. High affinity (20 pM) and low background signal of the free probe supports the determination of dissociation constants of tight-binding as well as low affinity inhibitors. The calculated lowest Kd value that can be accurately determined with the method is 60 fM.We also introduce graphical presentation of the linearized Cheng-Prusoff equation and demonstrate multiple possibilities for its application (deciding upon the assay formats, calculation of the limits of Kd determination, etc.). The open toolbox (http://www.ut.ee/medchem/toolbox-fluorescence-probes) is available for creating the map of resolvable affinities if applying the competitive probes at defined assay conditions.

Development of Quantum Chemical Method to Calculate Half Maximal Inhibitory Concentration (IC50 ).

Till date theoretical calculation of the half maximal inhibitory concentration (IC50 ) of a compound is based on different Quantitative Structure Activity Relationship (QSAR) models which are empirical methods. By using the Cheng-Prusoff equation it may be possible to compute IC50 , but this will be computationally very expensive as it requires explicit calculation of binding free energy of an inhibitor with respective protein or enzyme. In this article, for the first time we report an ab initio method to compute IC50 of a compound based only on the inhibitor itself where the effect of the protein is reflected through a proportionality constant. By using basic enzyme inhibition kinetics and thermodynamic relations, we derive an expression of IC50 in terms of hydrophobicity, electric dipole moment (μ) and reactivity descriptor (ω) of an inhibitor. We implement this theory to compute IC50 of 15 HIV-1 capsid inhibitors and compared them with experimental results and available other QASR based empirical results. Calculated values using our method are in very good agreement with the experimental values compared to the values calculated using other methods.

A microarray-based approach to evaluate the functional significance of protein-binding motifs

Intracellular proteins comprise numerous peptide motifs that interact with protein-binding domains. However, using sequence information alone, the identification of functionally relevant interaction motifs remains a challenge. Here, we present a microarray-based approach for the evaluation of peptides as protein-binding motifs. To this end, peptides corresponding to protein interaction motifs were spotted as a microarray. First, peptides were titrated with a pan-specific binder and the apparent Kd value of this binder for each peptide was determined. For phosphotyrosine-containing peptides, an anti-phosphotyrosine antibody was employed. Then, in the presence of the pan-specific binder, arrays were competitively titrated with cell lysate and competition constants were determined. Using the Cheng-Prusoff equation, binding constants for the pan-specific binder and inhibition constants for the lysates were converted into affinity constants for the lysate. We experimentally validate this method using a phosphotyrosine-binding SH2 domain as a further reference. Furthermore, strong binders correlated with binding motifs engaging in numerous interactions as predicted by Scansite. This method provides a highly parallel and robust approach to identify peptides corresponding to interaction motifs with strong binding capacity for proteins in the cell lysate.Graphical Using an antibody as a pan-specific binder the capacity of interaction motifs to bind to proteins from cell lysates can be probed. Competition of the antibody is observed for only those peptides to which a lysate protein bindsElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9382-6) contains supplementary material, which is available to authorized users.

Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities.

Glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase (GR) are metabolically quite important enzymes. Within this study, these two enzymes were purified for the first time from the gills of Lake Van fish. In the purifying process, ammonium sulfate precipitation and 2',5'-ADP Sepharose 4B affinity column chromatography techniques for glucose-6-phosphate dehydrogenase, temperature degradation and 2',5'-ADP Sepharose 4B affinity column chromatography for glutathione reductase enzyme were used. The control of the enzyme purity and determination of molecular weight were done with sodium dodecyl sulfate polyacrylamide gel electrophoresis. K(M) and V(max) values were determined with Lineweaver-Burk plot. Besides, the effects of some chalcone derivatives on the purified enzymes were analyzed. For the ones showing inhibition effect, % activity-[I] figures were drawn and IC50 values were determined. K(i) value was calculated by using Cheng-Prusoff equation.

Enzyme kinetics and inhibition of histone acetyltransferase KAT8

Lysine acetyltransferase 8 (KAT8) is a histone acetyltransferase (HAT) responsible for acetylating lysine 16 on histone H4 (H4K16) and plays a role in cell cycle progression as well as acetylation of the tumor suppressor protein p53. Further studies on its biological function and drug discovery initiatives will benefit from the development of small molecule inhibitors for this enzyme. As a first step towards this aim we investigated the enzyme kinetics of this bi-substrate enzyme. The kinetic experiments indicate a ping-pong mechanism in which the enzyme binds Ac-CoA first, followed by binding of the histone substrate. This mechanism is supported by affinity measurements of both substrates using isothermal titration calorimetry (ITC). Using this information, the KAT8 inhibition of a focused compound collection around the non-selective HAT inhibitor anacardic acid has been investigated. Kinetic studies with anacardic acid were performed, based on which a model for the catalytic activity of KAT8 and the inhibitory action of anacardic acid (AA) was proposed. This enabled the calculation of the inhibition constant Ki of anacardic acid derivatives using an adaptation of the Cheng–Prusoff equation. The results described in this study give insight into the catalytic mechanism of KAT8 and present the first well-characterized small-molecule inhibitors for this HAT.

Size dependence of steric shielding and multivalency effects for globular binding inhibitors.

Competitive binding inhibitors based on multivalent nanoparticles have shown great potential for preventing virus infections. However, general design principles of highly efficient inhibitors are lacking as the quantitative impact of factors such as virus concentration, inhibitor size, steric shielding, or multivalency effects in the inhibition process is not known. Based on two complementary experimental inhibition assays we determined size-dependent steric shielding and multivalency effects. This allowed us to adapt the Cheng-Prusoff equation for its application to multivalent systems. Our results show that the particle and volume normalized IC50 value of an inhibitor at very low virus concentration predominantly depends on its multivalent association constant, which itself exponentially increases with the inhibitor/virus contact area and ligand density. Compared to multivalency effects, the contribution of steric shielding to the IC50 values is only minor, and its impact is only noticeable if the multivalent dissociation constant is far below the virus concentration, which means if all inhibitors are bound to the virus. The dependence of the predominant effect, either steric shielding or multivalency, on the virus concentration has significant implications on the in vitro testing of competitive binding inhibitors and determines optimal inhibitor diameters for the efficient inhibition of viruses.

Crystal Structure and Pharmacological Characterization of a Novel N-Methyl-d-aspartate (NMDA) Receptor Antagonist at the GluN1 Glycine Binding Site

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with Kb values of 21-63 nM at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed >100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.

A New Radioligand Binding Assay to Measure the Concentration of Drugs in Rodent Brain Ex Vivo

We have developed a new radioligand binding assay method to measure the concentration of nonradiolabeled drugs in the brain ex vivo. This new method fuses the concepts of standard competition and saturation binding assays and uses a transformed version of the Cheng-Prusoff equation (Biochem Pharmacol 22:3099-3108, 1973) to calculate the drug concentration. After testing the validity of this method, we demonstrated its utility by measuring the brain concentration of sazetidine-A, a newly developed nicotinic receptor ligand, and its elimination rate after a single subcutaneous administration. Our results indicate that sazetidine-A reaches brain concentrations that are known to occupy and desensitize the majority of neuronal nicotinic acetylcholine receptor binding sites. Furthermore, using this method, we estimated the half-life of sazetidine-A in the rat brain to be ∼65 min. It is important to note that the method described here to measure sazetidine-A in brain should be generalizable to other drugs acting at any receptor that can be reliably measured with a radiolabeled ligand.

The in Vivo Toxicity of Hydroxyurea Depends on Its Direct Target Catalase

Hydroxyurea (HU) is a well tolerated ribonucleotide reductase inhibitor effective in HIV, sickle cell disease, and blood cancer therapy. Despite a positive initial response, however, most treated cancers eventually progress due to development of HU resistance. Although RNR properties influence HU resistance in cell lines, the mechanisms underlying cancer HU resistance in vivo remain unclear. To address this issue, we screened for HU resistance in the plant Arabidopsis thaliana and identified seventeen unique catalase mutants, thereby establishing that HU toxicity depends on catalase in vivo. We further demonstrated that catalase is a direct HU target by showing that HU acts as a competitive inhibitor of catalase-mediated hydrogen peroxide decomposition. Considering also that catalase can accelerate HU decomposition in vitro and that co-treatment with another catalase inhibitor alleviates HU effects in vivo, our findings suggests that HU could act as a catalase-activated pro-drug. Clinically, we found high catalase activity in circulating cells from untreated chronic myeloid leukemia, offering a possible explanation for the efficacy of HU against this malignancy.

Recognition Versus Adaptive Up-regulation and Degradation of CC Chemokines by the Chemokine Decoy Receptor D6 Are Determined by Their N-terminal Sequence

The chemokine decoy receptor D6 controls inflammatory responses by selective recognition and degradation of most CCR1 to CCR5 agonistic ligands. CCL14 is a homeostatic chemokine present at high concentrations in the serum with a weak agonist activity on CCR1. Under inflammatory conditions, plasmin and UPA-mediated truncation of 8 amino acids generates the potent CCR1/CCR3/CCR5 isoform CCL14(9-74), which is further processed and inactivated by dipeptidyl peptidase IV/CD26 that generates CCL14(11-74). Here we report that D6 efficiently binds both CCL14 and its truncated isoforms. Like other D6 ligands, the biologically active CCL14(9-74) induces adaptive up-regulation of D6 expression on the cell membrane and is rapidly and efficiently degraded. In contrast, the D6-mediated degradation of the biologically inactive isoforms CCL14(1-74) and CCL14(11-74) is very inefficient. Thus, D6 cooperates with CD26 in the negative regulation of CCL14 by the selective degradation of its biologically active isoform. Analysis of a panel of CC chemokines and their truncated isoforms revealed that D6-mediated chemokine degradation does not correlate with binding affinity. Conversely, degradation efficiency is positively correlated with D6 adaptive up-regulation. Sequence analysis indicated that a proline residue in position 2 of D6 ligands is dispensable for binding but crucial for D6 adaptive up-regulation and efficient degradation.

Identification of a Domain which Affects Kinetics and Antagonistic Potency of Clozapine at 5-HT3 Receptors

The widely used atypical antipsychotic clozapine is a potent competitive antagonist at 5-HT3 receptors which may contribute to its unique psychopharmacological profile. Clozapine binds to 5-HT3 receptors of various species. However, the structural requirements of the respective binding site for clozapine remain to be determined. Differences in the primary sequences within the 5-HT3A receptor gene in schizophrenic patients may result in an alteration of the antipsychotic potency and/or the side effect profile of clozapine. To determine these structural requirements we constructed chimeras with different 5-HT3A receptor sequences of murine and human origin and expressed these mutants in human embryonic kidney (HEK) 293 cells. Clozapine antagonises recombinant mouse 5-HT3A receptors with higher potency compared to recombinant human 5-HT3A receptors. 5-HT activation curves and clozapine inhibition curves yielded the parameters EC50 and IC50 for all receptors tested in the range of 0.6–2.7 µM and 1.5–83.3 nM, respectively. The use of the Cheng-Prusoff equation to calculate the dissociation constant Kb values for clozapine revealed that an extracellular sequence (length 86 aa) close to the transmembrane domain M1 strongly determines the binding affinity of clozapine. Kb values of clozapine were significantly lower (0.3–1.1 nM) for receptors containing the murine sequence and higher when compared with receptors containing the respective human sequence (5.8–13.4 nM). Thus, individual differences in the primary sequence of 5-HT3 receptors may be crucial for the antipsychotic potency and/or the side effect profile of clozapine.

The X-ray Structure of RU486 Bound to the Progesterone Receptor in a Destabilized Agonistic Conformation

Here we describe the 1.95 A structure of the clinically used antiprogestin RU486 (mifepristone) in complex with the progesterone receptor (PR). The structure was obtained by taking a crystal of the PR ligand binding domain containing the agonist norethindrone and soaking it in a solution containing the antagonist RU486 for extended times. Clear ligand exchange could be observed in one copy of the PR ligand binding domain dimer in the crystal. RU486 binds while PR is in an agonistic conformation without displacing helix 12. Although this is probably because of the constraints of the crystal lattice, it demonstrates that helix 12 displacement is not a prerequisite for RU486 binding. Interestingly, B-factor analysis clearly shows that helix 12 becomes more flexible after RU486 binding, suggesting that RU486, being a model antagonist, does not induce one fixed conformation of helix 12 but changes its positional equilibrium. This conclusion is confirmed by comparing the structures of RU486 bound to PR and RU486 bound to the glucocorticoid receptor.

Small Molecules Can Selectively Inhibit Ephrin Binding to the EphA4 and EphA2 Receptors

The erythropoietin-producing hepatocellular (Eph) family of receptor tyrosine kinases regulates a multitude of physiological and pathological processes. Despite the numerous possible research and therapeutic applications of agents capable of modulating Eph receptor function, no small molecule inhibitors targeting the extracellular domain of these receptors have been identified. We have performed a high throughput screen to search for small molecules that inhibit ligand binding to the extracellular domain of the EphA4 receptor. This yielded a 2,5-dimethylpyrrolyl benzoic acid derivative able to inhibit the interaction of EphA4 with a peptide ligand as well as the natural ephrin ligands. Evaluation of a series of analogs identified an isomer with similar inhibitory properties and other less potent compounds. The two isomeric compounds act as competitive inhibitors, suggesting that they target the high affinity ligand-binding pocket of EphA4 and inhibit ephrin-A5 binding to EphA4 with K(i) values of 7 and 9 mum in enzyme-linked immunosorbent assays. Interestingly, despite the ability of each ephrin ligand to promiscuously bind many Eph receptors, the two compounds selectively target EphA4 and the closely related EphA2 receptor. The compounds also inhibit ephrin-induced phosphorylation of EphA4 and EphA2 in cells, without affecting cell viability or the phosphorylation of other receptor tyrosine kinases. Furthermore, the compounds inhibit EphA4-mediated growth cone collapse in retinal explants and EphA2-dependent retraction of the cell periphery in prostate cancer cells. These data demonstrate that the Eph receptor-ephrin interface can be targeted by inhibitory small molecules and suggest that the two compounds identified will be useful to discriminate the activities of EphA4 and EphA2 from those of other co-expressed Eph receptors that are activated by the same ephrin ligands. Furthermore, the newly identified inhibitors represent possible leads for the development of therapies to treat pathologies in which EphA4 and EphA2 are involved, including nerve injuries and cancer.

A Novel Method for Determination of the Affinity of Protein: Protein Interactions in Homogeneous Assays

Nonradioactive homogeneous assays are widely used to screen for inhibitors of biomolecular interactions. To ensure optimal sensitivity for the detection of competitive inhibitors, reagent concentrations should be fixed at or below the K(D) of the protein-protein interaction. Accurate measurement of K(D) during assay development is therefore critical. Although conventional methods work well with heterogeneous assays, they are generally unsatisfactory with homogeneous systems. Here the authors describe an alternative method to determine the K(D) of protein-protein interactions in homogeneous assays. The method uses a rearrangement of the Cheng-Prusoff equation: IC50= (([Ki]/K(D)) x [L]) + Ki. A competitive inhibitor is titrated into the ligand-receptor binding assay at a range of ligand concentrations and IC50 values are calculated. Plotting measured IC50 versus concentration of ligand gives a linear plot with y-intercept (Ki) and gradient (Ki/K(D)). K(D) is the affinity constant for the ligand-receptor interaction. Here the authors use homogeneous time-resolved fluorescence (HTRF) in 2 model systems (TRAIL/TRAIL receptor 4 and OX40 ligand/OX40 receptor) and demonstrate that measured K(D) values calculated using the linearized Cheng-Prusoff plot compare favorably with those from independent experiments. The advantages and limitations of the method are discussed.