Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor

ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.     

Universal screening methods and applications of ThermoFluor

The genomics revolution has unveiled a wealth of poorly characterized proteins. Scientists are often able to produce milligram quantities of proteins for which function is unknown or hypothetical, based only on very distant sequence homology. Broadly applicable tools for functional characterization are essential to the illumination of these orphan proteins. An additional challenge is the direct detection of inhibitors of protein-protein interactions (and allosteric effectors). Both of these research problems are relevant to, among other things, the challenge of finding and validating new protein targets for drug action. Screening collections of small molecules has long been used in the pharmaceutical industry as 1 method of discovering drug leads. Screening in this context typically involves a function-based assay. Given a sufficient quantity of a protein of interest, significant effort may still be required for functional characterization, assay development, and assay configuration for screening. Increasingly, techniques are being reported that facilitate screening for specific ligands for a protein of unknown function. Such techniques also allow for function-independent screening with better characterized proteins. ThermoFluor, a screening instrument based on monitoring ligand effects on temperature-dependent protein unfolding, can be applied when protein function is unknown. This technology has proven useful in the decryption of an essential bacterial enzyme and in the discovery of a series of inhibitors of a cancer-related, protein-protein interaction. The authors review some of the tools relevant to these research problems in drug discovery, and describe our experiences with 2 different proteins.     

Screening and characterization of human monoglyceride lipase active site inhibitors using orthogonal binding and functional assays

Endocannabinoids such as 2-arachidonylglycerol (2-AG) are ligands for cannabinoid receptors that contribute to the transmission and modulation of pain signals. The antinociceptive effect of exogenous 2-AG suggests that inhibition of monoglyceride lipase (MGLL), the enzyme responsible for degrading 2-AG and arresting signaling, may be a target for pain modulation. Here we describe the characterization of MGLL ligands following a high-throughput screening campaign. Ligands were discovered using ThermoFluor, a label-free affinity-based screening tool that measures ligand binding via modulation of protein thermal stability. A kinetic fluorescent assay using the substrate 4-methylcoumarin butyrate was used to counterscreen confirmed HTS positives. A comparison of results from binding and inhibition assays allowed elucidation of compound mechanism of action. We demonstrate the limit of each technology and the benefits of using orthogonal assay techniques in profiling compounds.     

A strategy for high-throughput assay development using leads derived from nuclear magnetic resonance-based screening

A strategy is described for the development of high-throughput screening assays against targets of unknown function that involves the use of nuclear magnetic resonance (NMR) spectroscopy. Using this approach, molecules that bind to the protein target are identified from an NMR-based screen of a library of substrates, cofactors, and other compounds that are known to bind to many proteins and enzymes. Once a ligand has been discovered, a fluorescent or radiolabeled analog of the ligand is synthesized that can be used in a high-throughput screen. The approach is illustrated in the development of a high-throughput screening assay against HI-0033, a conserved protein from Haemophilus influenzae whose function is currently unknown. Adenosine was found to bind to HI-0033 by NMR, and fluorescent analogs were rapidly identified that bound to HI-0033 in the submicromolar range. Using these fluorescent compounds, a fluorescence polarization assay was developed that is suitable for high-throughput screening and obtaining detailed structure-activity relationships for lead optimization.     

Assay development and data analysis of receptor-ligand binding based on scintillation proximity assay

In this paper, we described the optimization of a generic binding assay to measure ligand-receptor interactions for peroxisome proliferator-activated receptors (PPARs). The assay is based on scintillation proximity assay, in which a protein is coated on scintillant-incorporated beads, and a radiolabeled ligand stimulates the beads to emit a signal by binding to the immobilized protein. An intrinsic binding affinity of unlabeled ligands is determined by competitive displacement of the radioligand. The protein coating and ligand binding are achieved in one step by simply mixing ligands, protein and beads in sequence. No additional steps of pre-coating and washing of beads are required. Protein is captured on beads effectively by electrostatic interactions, thus no affinity labeling of protein is required. In data analysis, ligands are grouped into two classes based on their binding affinities. For tight binding ligands, an equation is derived to accurately determine the binding affinity. Otherwise a general equation applies. This quantitative and high throughput assay provides a tool to screen a large library of molecules in search of potent ligands.     

Affinity ligand selection from a library of small molecules: assay development, screening, and application

A facile and cost-effective process for screening synthetic libraries for an affinity ligand is described. A high throughput 96-well plate filtration method was designed to screen both discrete compounds and mixtures of compounds attached to a solid support. Human serum albumin (HSA) was used as a target protein to demonstrate the proof of concept. Detection and quantitation by fluorescence was accomplished with the use of fluorescamine to conjugate the protein in the filtrate. It is found that mixtures demonstrating low average binding reflect an overall lower hit rate of the components, whereas deconvolution of mixtures with high protein binding consistently provides a high hit rate. This differs from many of the previous experiences screening solid-phase mixtures in which high false positive rates are noted to occur. A total of 100K compounds were tested: 25K as discrete samples and 75K as mixtures. An overall hit rate of 8% was observed. Secondary screening of compounds measured specificity, recovery, and dynamic binding capacity. The effectiveness of the method is illustrated using an affinity column made with a representative lead compound. A similar purity was achieved in a single-step purification of HSA from serum as compared to that obtained by two steps of ion-exchange chromatography. The process for primary screening of a large number of compounds is simple, inexpensive, and applicable to any soluble target protein of known or unknown function from crude mixtures and may have additional utility as a generic chemical affinity tool for the functional characterization of novel proteins emerging from proteomics work.     

Intrinsic affinity of protein – ligand binding by differential scanning calorimetry

Differential scanning calorimetry (DSC) determines the enthalpy change upon protein unfolding and the melting temperature of the protein. Performing DSC of a protein in the presence of increasing concentrations of specifically-binding ligand yields a series of curves that can be fit to obtain the protein–ligand dissociation constant as done in the fluorescence-based thermal shift assay (FTSA, ThermoFluor, DSF). The enthalpy of unfolding, as directly determined by DSC, helps improving the precision of the fit. If the ligand binding is linked to protonation reactions, the intrinsic binding constant can be determined by performing the affinity determination at a series of pH values. Here, the intrinsic, pH-independent, affinity of acetazolamide binding to carbonic anhydrase (CA) II was determined. A series of high-affinity ligands binding to CAIX, an anticancer drug target, and CAII showed recognition and selectivity for the anticancer isozyme. Performing the DSC experiment in buffers of highly different enthalpies of protonation enabled to observe the ligand unbinding-linked protonation reactions and estimate the intrinsic enthalpy of binding. The heat capacity of combined unfolding and unbinding was determined by varying the ligand concentrations. Taken together, these parameters provided a detailed thermodynamic picture of the linked ligand binding and protein unfolding process.     

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.     

Characterization of the heme in human cystathionine beta-synthase by X-ray absorption and electron paramagnetic resonance spectroscopies

Human cystathionine beta-synthase is one of two key enzymes involved in intracellular metabolism of homocysteine. It catalyzes a beta-replacement reaction in which the thiolate of homocysteine replaces the hydroxyl group of serine to give the product, cystathionine. The enzyme is unusual in its dependence on two cofactors: pyridoxal phosphate and heme. The requirement for pyridoxal phosphate is expected on the basis of the nature of the condensation reaction that is catalyzed; however the function of the heme in this protein is unknown. We have examined the spectroscopic properties of the heme in order to assign the axial ligands provided by the protein. The heme Soret peak of ferric cystathionine beta-synthase is at 428 nm and shifts to approximately 395 nm upon addition of the thiol chelator, mercuric chloride. This is indicative of 6-coordinate low-spin heme converting to a 5-coordinate high-spin heme. The enzyme as isolated exhibits a rhombic EPR signal with g values of 2.5, 2.3, and 1.86, which are similar to those of heme proteins and model complexes with imidazole/thiolate ligands. Mercuric chloride treatment of the enzyme results in conversion of the rhombic EPR signal to a g = 6 signal, consistent with formation of the high-spin ferric heme. The X-ray absorption data reveal that iron in ferric cystathionine beta-synthase is 6-coordinate, with 1 high-Z scatterer and 5 low-Z scatterers. This is consistent with the presence of 5 nitrogens and 1 sulfur ligand. Together, these data support assignment of the axial ligands as cysteinate and imidazole in ferric cystathionine beta-synthase.     

The expression of recombinant human LOX-1 and identifying its mimic ligands by fluorescence polarization-based high throughput screening

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was identified as a major receptor for oxidized low-density lipoprotein (oxLDL) in endothelial cells. LOX-1 critically mediates the endothelial dysfunction and the progression of atherosclerosis by oxLDL stimulation. It might be an important target for vascular endothelium. In order to obtain human LOX-1 and identify its mimic ligand for facilitating the study of LOX-1 function, a recombinant plasmid pPIC9K-His-hLOX-1 was structured and expressed human LOX-1 in Pichia pastoris GS115. Western blot analysis ensured the expressed recombinant human LOX-1 protein and a receptor-ligand binding assay showed that it had high binding affinity with oxLDL. With this receptor protein, a competitive fluorescence polarization-based high throughput screening method was established in a 384-well microplate to isolate the mimic ligands of human LOX-1. The evaluating parameter Z' value of 0.72 for this method showed that fluorescence polarization-based high throughput screening assay was robust and the results had a high reliability. By the fluorescence polarization-based high throughput screening assay, a total of 20,316 chemicals were screened, and 2 chemicals were identified that they have a high affinity with human LOX-1. Competitive uptake DiI-oxLDL assay by human LOX-1 transfected CHO-K1 cells further confirmed that two chemicals block the uptake of DiI-oxLDL. And the preliminary results indicated that isolated mimic ligands may act as a function of antagonist. The discovery of human LOX-1 mimic ligand would benefit to further study the function of LOX-1 and identify a novel avenue for prevention and treatment atherosclerosis.     

Binding similarity network of ligand

The protein and ligand interaction takes an important part in protein function. Both ligand and its binding site are essential components for understanding how the protein-ligand complex functions. Until now, there have been many studies about protein function and evolution, but they usually lacked ligand information. Accordingly, in this study, we tried to answer the following questions: how much ligand and binding site are associated with protein function, and how ligands themselves are related to each other in terms of binding site. To answer the questions, we presented binding similarity network of ligand. Through the network analysis, we attempted to reveal systematic relationship between the ligand and binding site. The results showed that ligand binding site and function were closely related (conservation ratio, 81%). We also showed conservative tendency of function in line with ligand structure similarity with some exceptional cases. In addition, the binding similarity network of ligand revealed scale-free property to some degree like other biological networks. Since most nodes formed highly connected cluster, a clustering coefficient was very high compared with random. All the highly connected ligands (hubs) were involved in various functions forming large cluster and tended to act as a bridge between modular clusters in the network.     

Ligand specificity in the CRAL-TRIO protein family

Intracellular trafficking of hydrophobic ligands is often mediated by specific binding proteins. The CRAL-TRIO motif is common to several lipid binding proteins including the cellular retinaldehyde binding protein (CRALBP), the alpha-tocopherol transfer protein (alpha-TTP), yeast phosphatidylinositol transfer protein (Sec14p), and supernatant protein factor (SPF). To examine the ligand specificity of these proteins, we measured their affinity toward a variety of hydrophobic ligands using a competitive [(3)H]-RRR-alpha-tocopherol binding assay. Alpha-TTP preferentially bound RRR-alpha-tocopherol over all other tocols assayed, exhibiting a K(d) of 25 nM. Binding affinities of other tocols for alphaTTP closely paralleled their ability to inhibit in vitro intermembrane transfer and their potency in biological assays. All other homologous proteins studied bound alpha-tocopherol but with pronouncedly weaker (> 10-fold) affinities than alpha-TTP. Sec14p demonstrated a K(d) of 373 nM for alpha-tocopherol, similar to that for its native ligand, phosphatidylinositol (381 nM). Human SPF had the highest affinity for phosphatidylinositol (216 nM) and gamma-tocopherol (268 nM) and significantly weaker affinity for alpha-tocopherol (K(d) 615 nM). SPF bound [(3)H]-squalene more weakly (879 nM) than the other ligands. Our data suggest that of all known CRAL-TRIO proteins, only alphaTTP is likely to serve as the physiological mediator of alpha-tocopherol's biological activity. Further, ligand promiscuity observed within this family suggests that caution should be exercised when suggesting protein function(s) from measurements utilizing a single ligand.     

Identification of the ligands of protein interaction domains through a functional approach

The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (PTB) domains of human proteins. Of 47 PTB domains, we found that the overexpression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of RabGAP1, and EF1alpha, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays.     

Ligand based virtual screening and biological evaluation of inhibitors of chorismate mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv

We have identified new lead candidates that possess inhibitory activity against Mycobacterium tuberculosis H37Rv chorismate mutase by a ligand-based virtual screening optimized for lead evaluation in combination with in vitro enzymatic assay. The initial virtual screening using a ligand-based pharmacophore model identified 95 compounds from an in-house small molecule database of 15,452 compounds. The obtained hits were further evaluated by molecular docking and 15 compounds were short listed based on docking scores and the other scoring functions and subjected to biological assay. Chorismate mutase activity assays identified four compounds as inhibitors of M. tuberculosis chorismate mutase (MtCM) with low K(i) values. The structural models for these ligands in the chorismate mutase binding site will facilitate medicinal chemistry efforts for lead optimization against this protein.     

Determination of the volume changes induced by ligand binding to heat shock protein 90 using high-pressure denaturation

The volume changes accompanying ligand binding to proteins are thermodynamically important and could be used in the design of compounds with specific binding properties. Measuring the volumetric properties could yield as much information as the enthalpic properties of binding. Pressure-based methods are significantly more laborious than temperature methods and are underused. Here we present a pressure shift assay (PressureFluor, analogous to the ThermoFluor thermal shift assay) that uses high pressure to denature proteins. The PressureFluor method was used to study the ligand binding thermodynamics of heat shock protein 90 (Hsp90). Ligands stabilize the protein against pressure denaturation, similar to the stabilization against temperature denaturation. The equations that relate the ligand dosing, protein concentration, and binding constant with the volumes and compressibilities of unfolding and binding are presented.     

Detection of functional ligand-binding events using synchrotron x-ray scattering

Small-molecule ligands that change the structure of a protein are likely to affect its function, whereas those causing no structural change are less likely to be functional. Wide-angle x-ray scattering (WAXS) can be easily carried out on proteins and small molecules in solution in the absence of chemical tags or derivatives. The authors demonstrate that WAXS is a sensitive probe of ligand binding to proteins in solution and can distinguish between nonfunctional and productive binding. Furthermore, similar ligand-binding modes translate into similar scattering patterns. This approach has high potential as a novel, generic, low-throughput assay for functional ligand binding.     

Development of surface plasmon resonance biosensor assays for primary and secondary screening of acetylcholine binding protein ligands

Surface plasmon resonance (SPR) biosensors recently gained an important place in drug discovery. Here we present a primary and secondary SPR biosensor screening methodology. The primary screening method is based on a direct binding assay with covalent immobilized drug target proteins. For the secondary screening method, a sequential competition assay has been developed where the captured protein is first exposed to an unknown test compound, followed directly by an exposure to a high-molecular-weight reporter ligand. Using the high-molecular-weight reporter ligand to probe the remaining free binding site on the sensor, a significant signal enhancement is obtained. Furthermore, this assay format allows the validation of the primary direct binding assay format, efficiently revealing false positive data. As a model system, acetylcholine binding protein (AChBP), which is a soluble model protein for neuronal nicotinic acetylcholine receptors, has been used. The secondary assay is lower in throughput than the primary assay; however, the signal-to-noise ratio is two times higher compared with the direct assay, and it has a z′ factor of 0.96. Using both assays, we identified the compound tacrine as a ligand for AChBP.     

Structural and Functional Similarity between the Bacterial Type III Secretion System Needle Protein PrgI and the Eukaryotic Apoptosis Bcl-2 Proteins

Background

Functional similarity is challenging to identify when global sequence and structure similarity is low. Active-sites or functionally relevant regions are evolutionarily more stable relative to the remainder of a protein structure and provide an alternative means to identify potential functional similarity between proteins. We recently developed the FAST-NMR methodology to discover biochemical functions or functional hypotheses of proteins of unknown function by experimentally identifying ligand binding sites. FAST-NMR utilizes our CPASS software and database to assign a function based on a similarity in the structure and sequence of ligand binding sites between proteins of known and unknown function.

Methodology/Principal Findings

The PrgI protein from Salmonella typhimurium forms the needle complex in the type III secretion system (T3SS). A FAST-NMR screen identified a similarity between the ligand binding sites of PrgI and the Bcl-2 apoptosis protein Bcl-xL. These ligand binding sites correlate with known protein-protein binding interfaces required for oligomerization. Both proteins form membrane pores through this oligomerization to release effector proteins to stimulate cell death. Structural analysis indicates an overlap between the PrgI structure and the pore forming motif of Bcl-xL. A sequence alignment indicates conservation between the PrgI and Bcl-xL ligand binding sites and pore formation regions. This active-site similarity was then used to verify that chelerythrine, a known Bcl-xL inhibitor, also binds PrgI.

Conclusions/Significance

A structural and functional relationship between the bacterial T3SS and eukaryotic apoptosis was identified using our FAST-NMR ligand affinity screen in combination with a bioinformatic analysis based on our CPASS program. A similarity between PrgI and Bcl-xL is not readily apparent using traditional global sequence and structure analysis, but was only identified because of conservation in ligand binding sites. These results demonstrate the unique opportunity that ligand-binding sites provide for the identification of functional relationships when global sequence and structural information is limited.     

Using the protein chip to screen agonists and antagonists of the androgen receptor

Based on its important physical and pathological function, the androgen receptor (AR) is regarded as a significant drug target. In this report, the authors describe a novel strategy of protein chip technology to screen agonists and antagonists of AR. First, the AR ligand binding domain (AR-LBD) was expressed in Escherichia coli, purified, and then immobilized on a silane-polysaccharide surface of a protein chip. Second, the affinities of methyltestosterone (MT) and fluorescent-labeled testosterone for the AR-LBD protein chip were determined. Third, a converse strategy of the protein chip was tested to evaluate its reliability as a drug screening method. Fourth, a 10,067-compound library was screened to find new ligands of AR. From the results, the K(d) of testosterone and the IC(50) of MT are consistent with the literature (0.61 vs. 0.49 nM 2.88 vs. 3.90 nM, respectively). The Z' factor of the high-throughput screening (HTS) method was 0.76, which meets the requirement of drug screening (>0.4). Finally, 3 active ligands of AR were identified with their IC( 50) values of 3.63, 2.19, and 1.71 microM, respectively. In summary, the novel strategy of the AR-LBD protein chip was suitable for HTS at the molecular level.     

Structural proteomics: a tool for genome annotation

In any newly sequenced genome, 30% to 50% of genes encode proteins with unknown molecular or cellular function. Fortunately, structural genomics is emerging as a powerful approach of functional annotation. Because of recent developments in high-throughput technologies, ongoing structural genomics projects are generating new structures at an unprecedented rate. In the past year, structural studies have identified many new structural motifs involved in enzymatic catalysis or in binding ligands or other macromolecules (DNA, RNA, protein). The efficiency by which function is deduced from structure can be further improved by the integration of structure with bioinformatics and other experimental approaches, such as screening for enzymatic activity or ligand binding.