Identification of potent inhibitors of the sortilin-progranulin interaction.

High-throughput screening methods have been used to identify two novel series of inhibitors that disrupt progranulin binding to sortilin. Exploration of structure-activity relationships (SAR) resulted in compounds with sufficient potency and physicochemical properties to enable co-crystallization with sortilin. These co-crystal structures supported observed SAR trends and provided guidance for additional avenues for designing compounds with additional interactions within the binding site.

Structural characterization of nonactive site, TrkA-selective kinase inhibitors.

Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research of novel targets against pain. Although originally identified as an oncogene, Tropomyosin-related kinase A (TrkA) is linked to pain and elevated levels of NGF (the ligand for TrkA) are associated with chronic pain. Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief. Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases. The X-ray structures reveal a binding site outside the kinase active site that uses residues from the kinase domain and the juxtamembrane region. Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes. The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes. The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition. This finding highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information because of substantial differences in the interacting residues. These structures illustrate the flexibility of binding to sequences outside of-but adjacent to-the kinase domain of TrkA. This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins.

Application of affinity selection-mass spectrometry assays to purification and affinity-based screening of the chemokine receptor CXCR4.

Affinity selection-mass spectrometry (AS-MS) is a sensitive technology for identifying small molecules that bind to target proteins, and assays enabled by AS-MS can be used to delineate relative binding affinities of ligands for proteins. 'Indirect' AS-MS assays employ size-exclusion techniques to separate target-ligand complexes from unbound ligands, and target-associated ligands are then specifically detected by liquid chromatography mass spectrometry. We report how indirect AS-MS binding assays with known reference control compounds were used as guideposts for development of an optimized purification method for CXCR4, a G-protein coupled chemokine receptor, for which we sought novel antagonists. The CXCR4 purification method that was developed was amenable to scale-up and enabled the screening of purified recombinant human CXCR4 against a large combinatorial library of small molecules by high throughput indirect AS-MS. The screen resulted in the discovery of new ligands that competed off binding of reference compounds to CXCR4 in AS-MS binding assays and that antagonized SDF1α-triggered responses and CXCR4-mediated HIV1 viral uptake in cell-based assays. This report provides a methodological paradigm whereby indirect AS-MS-based ligand binding assays may be used to guide optimal integral membrane protein purification methods that enable downstream affinity selection-based applications such as high throughput AS-MS screens.

Multidimensional profiling of CSF1R screening hits and inhibitors: assessing cellular activity, target residence time, and selectivity in a higher throughput way.

Over the past years, improvements in high-throughput screening (HTS) technology and compound libraries have resulted in a dramatic increase in the amounts of good-quality screening hits, and there is a growing need for follow-on hit profiling assays with medium throughput to further triage hits. Here the authors present such assays for the colony-stimulating factor 1 receptor (CSF1R, Fms), including tests for cellular activity and a homogeneous assay to measure affinity for inactive CSF1R. They also present a high-throughput assay to measure target residence time, which is based on competitive binding kinetics. To better fit k(off) rates, they present a modified mathematical model for competitive kinetics. In all assays, they profiled eight reference inhibitors (imatinib, sorafenib, sunitinib, tandutinib, dasatinib, GW2580, Ki20227, and J&J's pyrido[2,3-d]pyrimidin-5-one). Using the known biochemical selectivities of these inhibitors, which can be quantified using metrics such as the selectivity entropy, the authors have determined which assay readout best predicts hit selectivity. Their profiling shows surprisingly that imatinib has a preference for the active form of CSF1R and that Ki20227 has an unusually slow target dissociation rate. This confirms that follow-on hit profiling is essential to ensure that the best hits are selected for lead optimization.

Evaluation of in vitro PXR-based assays and in silico modeling approaches for understanding the binding of a structurally diverse set of drugs to PXR.

The pregnane X-receptor (PXR) is a promiscuous nuclear receptor primarily responsible for the induction of genes from the cytochrome P450 3A family. In this study, we used a previously described PXR/SRC tethered protein to establish two in vitro assays for identifying PXR ligands: automated ligand identification system (ALIS) and temperature-dependent circular dichroism (TdCD). Kd values determined by ALIS and TdCD showed good correlations with the EC50 values determined by a PXR luciferase reporter-gene assay for 37 marketed drugs. The same set of compounds was modeled into the PXR ligand-binding domain that takes into consideration the structural variations of five published X-ray structures of PXR-ligand complexes. Major findings from our in silico analysis are as follows. First, the primary determinants for non-binders of PXR are molecular size and shape of the compounds. Low molecular weight (MW<300) compounds were in general found to be non-binders, and those molecules that do not match the shape of the PXR ligand-binding site may also act as a non-binder. Secondly, the favorable hydrophobic interactions, mostly through aromatic π-π interactions, and the presence of suitable hydrogen bond(s) between the compounds and PXR are attributes of strong binders. Thirdly, the structures of the PXR binding domain possess the flexibility that accommodates structurally diverse compounds, while some of the strong binders may also adapt flexible conformations for fitting into the binding site. The results from this study provide a molecular basis for future efforts in reducing/abolishing the PXR-dependent CYP3A4 induction liability.

[(3)H]chlorpromazine photolabeling of the torpedo nicotinic acetylcholine receptor identifies two state-dependent binding sites in the ion channel.

Chlorpromazine (CPZ), a potent nicotinic acetylcholine receptor (nAChR) noncompetitive antagonist, binds with higher affinity in the ion channel in the desensitized state than in the closed channel state and with low affinity to additional sites in nAChR-rich membranes. For nAChR equilibrated with agonist, we confirm previous reports that [(3)H]CPZ occupies a site near the cytoplasmic end of the M2 ion channel domain, photolabeling positions M2-2, M2-6, and/or M2-9 in each subunit. We find that [(3)H]CPZ also binds at the extracellular end of the channel, photolabeling amino acids at positions M2-16 (alpha,gamma), M2-17 (alpha,beta,delta), and M2-20 (alpha,beta,delta). The photolabeling at the cytoplasmic end of the channel is fully inhibitable by phencyclidine or proadifen, whereas neither drug inhibits [(3)H]CPZ photolabeling at the extracellular end, establishing that positively charged drugs can bind simultaneously at the cytoplasmic and extracellular ends of the ion channel. [(3)H]CPZ photolabeling is not detected in the transmembrane domain outside the ion channel, but it photolabels alphaMet-386 and alphaSer-393 in the cytoplasmic alphaMA helix. In the nAChR equilibrated with alpha-bungarotoxin to stabilize the nAChR in a closed state, [(3)H]CPZ photolabels amino acids at M2-5 (alpha), M2-6 (alpha,beta,delta), and M2-9 (beta,delta), with no labeling at M2-2. These results provide novel information about the modes of drug binding within the nAChR ion channel and indicate that within the nAChR transmembrane domain, the binding of cationic aromatic amine antagonists can be restricted to the ion channel domain, in contrast to the uncharged, allosteric potentiators and inhibitors that also bind within the delta subunit helix bundle and at subunit interfaces.

Affinity selection-mass spectrometry screening techniques for small molecule drug discovery.

Affinity selection-mass spectrometry (AS-MS) techniques assess the binding of candidate molecules to immobilized or soluble receptors, and these methods are gaining acceptance in high throughput screening laboratories as valuable complements to traditional drug discovery technologies. A diversity of receptor types have been evaluated by AS-MS, including those that are difficult to screen using traditional biochemical approaches. AS-MS techniques that couple liquid chromatography-MS with size-based separation methods, such as ultrafiltration, gel permeation, or size-exclusion chromatography, are particularly amenable to the demands of MS-based screening and have demonstrated the greatest success across a broad range of drug targets. MS measurements of receptor function have many of the same advantages as AS-MS screening and are increasingly used for drug discovery as well.

Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry.

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.

Photolabeling the Torpedo nicotinic acetylcholine receptor with 4-azido-2,3,5,6-tetrafluorobenzoylcholine, a partial agonist.

The interactions of a photoreactive analogue of benzoylcholine, 4-azido-2,3,5,6-tetrafluorobenzoylcholine (APFBzcholine), with nicotinic acetylcholine receptors (nAChRs) were studied using electrophysiology and photolabeling. APFBzcholine acted as a low-efficacy partial agonist, eliciting maximal responses that were 0.3 and 0.1% of that of acetylcholine for embryonic mouse and Torpedo nAChRs expressed in Xenopus oocytes, respectively. Equilibrium binding studies of [3H]APFBzcholine with nAChR-rich membranes from Torpedo electric organ revealed equal affinities (K(eq) = 12 microM) for the two agonist binding sites. Upon UV irradiation at 254 nm, [3H]APFBzcholine was photoincorporated into the nAChR alpha, gamma, and delta subunits in an agonist-inhibitable manner. Photolabeled amino acids in the agonist binding sites were identified by Edman degradation of isolated, labeled subunit fragments. [3H]APFBzcholine photolabeled gammaLeu-109/deltaLeu-111, gammaTyr-111, and gammaTyr-117 in binding site segment E as well as alphaTyr-198 in alpha subunit binding site segment C. The observed pattern of photolabeling is examined in relation to the predicted orientation of the azide when APFBzcholine is docked in the agonist binding site of a homology model of the nAChR extracellular domain based upon the structure of the snail acetylcholine binding protein.

Identification of binding sites in the nicotinic acetylcholine receptor for [3H]azietomidate, a photoactivatable general anesthetic.

To identify binding domains in a ligand-gated ion channel for etomidate, an intravenous general anesthetic, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with a photoactivatable analog, [(3)H]azietomidate. Based upon the inhibition of binding of the noncompetitive antagonist [(3)H]phencyclidine, azietomidate and etomidate bind with 10-fold higher affinity to nAChRs in the desensitized state (IC(50) = 70 microm) than in the closed channel state. In addition, both drugs between 0.1 and 1 mm produced a concentration-dependent enhancement of [(3)H]ACh equilibrium binding affinity, but they inhibited binding at higher concentrations. UV irradiation resulted in preferential [(3)H]azietomidate photoincorporation into the nAChR alpha and delta subunits. Photolabeled amino acids in both subunits were identified in the ion channel domain and in the ACh binding sites by Edman degradation. Within the nAChR ion channel in the desensitized state, there was labeling of alphaGlu-262 and deltaGln-276 at the extracellular end and deltaSer-258 and deltaSer-262 toward the cytoplasmic end. Within the acetylcholine binding sites, [(3)H]azietomidate photolabeled alphaTyr-93, alphaTyr-190, and alphaTyr-198 in the site at the alpha-gamma interface and deltaAsp-59 (but not the homologous position, gammaGlu-57). Increasing [(3)H]azietomidate concentration from 1.8 to 150 microm increased the efficiency of incorporation into amino acids within the ion channel by 10-fold and in the ACh sites by 100-fold, consistent with higher affinity binding within the ion channel. The state dependence and subunit selectivity of [(3)H]azietomidate photolabeling are discussed in terms of the structures of the nAChR transmembrane and extracellular domains.

Interactions of peptide mimics of hyaluronic acid with the receptor for hyaluronan mediated motility (RHAMM).

Using the hyaluronic acid (HA) binding region of the receptor for hyaluronan-mediated motility (RHAMM) as a model, a molecular perspective for peptide mimicry of the natural ligand was established by comparing the interaction sites of HA and unnatural peptide-ligands to RHAMM. This was accomplished by obtaining a series of octapeptide-ligands through screening experiments that bound to the HA binding domains of RHAMM (amino acids 517-576) and could be displaced by HA. These molecules were computationally docked onto a three-dimensional NMR based model of RHAMM. The NMR model showed that RHAMM(517-576) was a set of three helices, two of which contained the HA binding domains (HABDs) flanking a central groove. The structure was stabilized by hydrophobic interactions from four pairs of Val and Ile side chains extending into the groove. The presence of solvent exposed, positively charged side chains spaced 11 A apart matched the spacing of negative charges on HA. Docking experiments using flexible natural and artificial ligands demonstrated that HA and peptide-mimetics preferentially bound to the second helix that contains HABD-2. Three salt bridges between HA carboxylates and Lys548, Lys553 and Lys560 and two hydrophobic interactions involving Val538 and Val559 were predicted to stabilize the RHAMM-HA complex. The high affinity peptides and HA utilized the same charged residues, with additional contacts to other basic residues. However, hydrophobic contacts do not contribute to affinity for peptide ligand-RHAMM complexes. These results offer insight into how selectivity is achieved in the binding of HA to RHAMM, and how peptide competitors may compete for binding with HA on a single hyaladherin.

2-(3-Methyl-3H-diaziren-3-yl)ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate: a derivative of the stereoselective general anesthetic etomidate for photolabeling ligand-gated ion channels.

To locate general anesthetic binding sites on ligand-gated ion channels, a diazirine derivative of the potent intravenous anesthetic, R-(+)-etomidate (2-ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate), has been synthesized and characterized. R-(+)-Azietomidate [2-(3-methyl-3H-diaziren-3-yl)ethyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate] anesthetizes tadpoles with an EC(50) of 2.2 microM, identical to that of R-(+)-etomidate. At this concentration both agents equally enhanced GABA-induced currents and decreased binding of the caged-convulsant [(35)S]TBPS to GABA(A) receptors. In all of the above actions R-(+)-azietomidate is about an order of magnitude more potent than S-(-)-azietomidate, an enantioselectivity comparable to etomidate's. R-(+)-Azietomidate also inhibits acetylcholine-induced currents in nicotinic acetylcholine receptors, with about twice the potency of the parent compound. [(3)H]Azietomidate photoincorporated into Torpedo nicotinic acetylcholine receptor-rich membranes. Desensitization decreased photoincorporation into the delta-subunit and increased that into the alpha-subunit. The latter increase was confined to a proteolytic fragment containing the first three transmembrane segments. Thus, R-(+)-azietomidate is a potent stereoselective general anesthetic and an effective photolabel.

Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine, a novel photoaffinity antagonist.

[(3)H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (K(D) approximately 10 microM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [(3)H]TDBzcholine incorporate (3)H into the alpha, gamma, and delta subunits in an agonist-inhibitable manner. The specific residues labeled by [(3)H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the alpha subunit, [(3)H]TDBzcholine photoincorporated into alphaCys-192, alphaCys-193, and alphaPro-194. For the gamma and delta subunits, [(3)H]TDBzcholine incorporated into homologous leucine residues, gammaLeu-109 and deltaLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the alpha subunit Segment C are oriented toward the agonist site and are in proximity to gammaLeu-109/deltaLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.