Fluorescent Thermal Shift Research Articles

Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX

BackgroundHuman carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding. MethodsThe observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry. ResultsThe pKa of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was −24kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01nM and the observed affinity of 2nM. ConclusionsThe intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship. General significanceIt is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations.

Thermodynamic characterization of human carbonic anhydrase VB stability and intrinsic binding of compounds

The thermodynamics of low molecular weight synthetic sulfonamide inhibitor binding to carbonic anhydrase (CA) VB was determined by the isothermal titration calorimetry (ITC) and the fluorescent thermal shift assay (FTSA). ITC provided the enthalpic and entropic contributions to the binding affinity of ethoxzolamide to CA VB. FTSA is a high-throughput assay that measures protein thermal stabilization by added ligands. FTSA enabled determination of extremely high affinity of several compounds binding to CA VB. CA VB is one of two isoforms that are expressed in mitochondria, participate in carbon metabolism and pH homeostasis and are implicated in diseases such as obesity. Therefore CA VB is a drug target. Here a series of para-substituted tetrafluoro benzenesulfonamides were investigated as high affinity inhibitors of CA VB. Thermodynamic equilibrium binding measurements such as ITC and FTSA provide only the observed parameters. Dissection of binding-linked reactions is necessary to obtain the intrinsic parameters that in turn could be correlated with the chemical structure of the inhibitors. Intrinsic dissociation constants of the inhibitors were estimated and they reached 1 pM, one of the strongest binding reactions observed between any protein–ligand binding.

Substrate-Induced Allosteric Change in the Quaternary Structure of the Spermidine N-Acetyltransferase SpeG

The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Two hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. Our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites.

Approved Drugs Containing Thiols as Inhibitors of Metallo-β-lactamases: Strategy To Combat Multidrug-Resistant Bacteria.

Resistance to β-lactam antibiotics can be mediated by metallo-β-lactamase enzymes (MBLs). An MBL inhibitor could restore the effectiveness of β-lactams. We report on the evaluation of approved thiol-containing drugs as inhibitors of NDM-1, VIM-1, and IMP-7. Drugs were assessed by a novel assay using a purchasable fluorescent substrate and thermal shift. Best compounds were tested in antimicrobial susceptibility assay. Using these orthogonal screening methods, we identified drugs that restored the activity of imipenem.

A combinatorial biophysical approach; FTSA and SPR for identifying small molecule ligands and PAINs

Biophysical methods have emerged as attractive screening techniques in drug discovery both as primary hit finding methodologies, as in the case of weakly active compounds such as fragments, and as orthogonal methods for hit validation for compounds discovered through conventional biochemical or cellular assays. Here we describe a dual method employing fluorescent thermal shift assay (FTSA), also known as differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR), to interrogate ligands of the kinase p38α as well as several known pan-assay interference compounds (PAINs) such as aggregators, redox cyclers, and fluorescence quenchers. This combinatorial approach allows for independent verification of several biophysical parameters such as KD, kon, koff, ΔG, ΔS, and ΔH, which may further guide chemical development of a ligand series. Affinity values obtained from FTSA curves allow for insight into compound binding compared with reporting shifts in melting temperature. Ligand–p38 interaction data were in good agreement with previous literature. Aggregators and fluorescence quenchers appeared to reduce fluorescence signal in the FTSAs, causing artificially high shifts in Tm values, whereas redox compounds caused either shifts in affinity that did not agree between FTSA and SPR or a depression of FTSA signal.

Functionalization of fluorinated benzenesulfonamides and their inhibitory properties toward carbonic anhydrases.

Substituted tri- and tetrafluorobenzenesulfonamides were designed, synthesized, and evaluated as high-affinity and isoform-selective carbonic anhydrase (CA) inhibitors. Their binding affinities for recombinant human CA I, II, VA, VI, VII, XII, and XIII catalytic domains were determined by fluorescent thermal shift assay, isothermal titration calorimetry, and a stopped-flow CO2 hydration assay. Variation of the substituents at the 2-, 3-, and 4-positions yielded compounds with a broad range of binding affinities and isoform selectivities. Several 2,4-substituted-3,5,6-trifluorobenzenesulfonamides were effective CA XIII inhibitors with high selectivity over off-target CA I and CA II. 3,4-Disubstituted-2,5,6-trifluorobenzenesulfonamides bound CAs with higher affinity than 2,4-disubstituted-3,5,6-trifluorobenzenesulfonamides. Many such fluorinated benzenesulfonamides were found to be nanomolar inhibitors of CA II, CA VII, tumor-associated CA IX and CA XII, and CA XIII. X-ray crystal structures of inhibitors bound in the active sites of several CA isoforms provide structure-activity relationship information for inhibitor binding affinities and selectivity.

Intrinsic thermodynamics of trifluoromethanesulfonamide and ethoxzolamide binding to human carbonic anhydrase VII.

Human carbonic anhydrase (CA) isozyme VII is a cytosolic protein that is highly expressed in the cortex, hippocampus, and thalamus regions within mammalian brain, and expression disorders can cause epilepsy and several cases of malignant brain tumors. Therefore, CA VII is a potential antiepileptic and anticancer drug target. There are numerous sulfonamides that target CAs nonspecifically. It is important to understand the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex in order to design specific inhibitors against CA VII. Isothermal titration calorimetry and fluorescent thermal shift assay were used to characterize the intrinsic thermodynamic parameters of trifluoromethanesulfonamide and ethoxzolamide binding to CA VII. Binding experiments were carried out at various pH in different buffers in order to dissect linked protonation of the water molecule bound to the CA VII active site, deprotonation of the sulfonamide group of the inhibitor, and protonation-deprotonation of buffer. Dissection of all those contributions yielded the intrinsic thermodynamic parameters of binding, such as Gibbs free energy, binding enthalpy, entropy, and protein pKa value. Thermal shift assay was also used to determine CA VII stability at various pH.

Intrinsic binding of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides to native and recombinant human carbonic anhydrase VI.

Carbonic anhydrase (CA) VI is a potential drug target for cariogenesis and cancer of the salivary gland. It is the only secreted human CA isozyme which is found in saliva and milk. Here, CA VI was expressed in bacterial and mammalian cell cultures and directly affinity-purified from human saliva. The binding of 4-substituted-2,3,5,6-tetrafluorobenezenesulfonamides to the native and recombinant CA VI from these three sources was compared. Interaction between the enzyme and inhibitors was determined by fluorescent thermal shift assay and isothermal titration calorimetry. The observed dissociation constants were the same within the error margin for all three CA VI preparations. The intrinsic binding parameters for the compounds were obtained by determining and dissecting the binding-linked protonation reactions. Intrinsic thermodynamic parameters of binding arrange the compounds in a buffer- and pH-independent manner. Intrinsic binding constants of nonfluorinated compounds were significantly stronger than those of fluorinated benzenesulfonamides. An opposite result was determined for the observed binding constants. The increase in observed affinity of the fluorinated compounds was due to the fluorine effect on diminishing the pKa of the compounds but not due to direct recognition of the protein. The temperature-stability profiles for recombinant and native CA VI were compared and showed that CA VI is more stable in slightly acidic than neutral conditions.

Intrinsic Thermodynamics - Structure Correlation of Carbonic Anhydrase Inhibitors

For the design of selective inhibitors towards 12 active human carbonic anhydrase (CA) isoforms, a map correlating thermodynamics of inhibitor binding with cocrystal structures was created. A series of over 600 inhibitors were chemically synthesized. Their binding to CA isoforms was measured by three biophysical and biochemical techniques, namely, isothermal titration calorimetry (ITC), fluorescent thermal shift assay (FTSA), and enzymatic inhibition methods. A database of over 3000 binding reactions, including the intrinsic Gibbs free energies and sometimes the enthalpies, entropies and heat capacities, was assembled. Intrinsic energetics of binding was determined by accounting for the linked protonation-deprotonation reactions of both protein and ligand. Together with over 80 cocrystal structures, this database provided insight into some general issues of protein-ligand interactions, especially the enthalpy - entropy compensation and isoform-selective ligand design. Parts of some ligands contained flexible tailgroups that correlated with increased intrinsic entropy of binding. All designed ligands exhibited enthalpy-driven binding with minor but important entropic contributions. The method is useful for the design of ligands with appropriate affinities and selectivities.

Intrinsic Thermodynamics and Structure Correlation of Benzenesulfonamides with a Pyrimidine Moiety Binding to Carbonic Anhydrases I, II, VII, XII, and XIII.

The early stage of drug discovery is often based on selecting the highest affinity lead compound. To this end the structural and energetic characterization of the binding reaction is important. The binding energetics can be resolved into enthalpic and entropic contributions to the binding Gibbs free energy. Most compound binding reactions are coupled to the absorption or release of protons by the protein or the compound. A distinction between the observed and intrinsic parameters of the binding energetics requires the dissection of the protonation/deprotonation processes. Since only the intrinsic parameters can be correlated with molecular structural perturbations associated with complex formation, it is these parameters that are required for rational drug design. Carbonic anhydrase (CA) isoforms are important therapeutic targets to treat a range of disorders including glaucoma, obesity, epilepsy, and cancer. For effective treatment isoform-specific inhibitors are needed. In this work we investigated the binding and protonation energetics of sixteen [(2-pyrimidinylthio)acetyl]benzenesulfonamide CA inhibitors using isothermal titration calorimetry and fluorescent thermal shift assay. The compounds were built by combining four sulfonamide headgroups with four tailgroups yielding 16 compounds. Their intrinsic binding thermodynamics showed the limitations of the functional group energetic additivity approach used in fragment-based drug design, especially at the level of enthalpies and entropies of binding. Combined with high resolution crystal structural data correlations were drawn between the chemical functional groups on selected inhibitors and intrinsic thermodynamic parameters of CA-inhibitor complex formation.

4-amino-substituted benzenesulfonamides as inhibitors of human carbonic anhydrases.

A series of N-aryl-β-alanine derivatives and diazobenzenesulfonamides containing aliphatic rings were designed, synthesized, and their binding to carbonic anhydrases (CA) I, II, VI, VII, XII, and XIII was studied by the fluorescent thermal shift assay and isothermal titration calorimetry. The results showed that 4-substituted diazobenzenesulfonamides were more potent CA binders than N-aryl-β-alanine derivatives. Most of the N-aryl-β-alanine derivatives showed better affinity for CA II while diazobenzenesulfonamides possessed nanomolar affinities towards CA I isozyme. X-ray crystallographic structures showed the modes of binding of both compound groups.

Structural characterization of a hypothetical protein: a potential agent involved in trimethylamine metabolism in Catenulispora acidiphila

Catenulispora acidiphila is a newly identified lineage of actinomycetes that produces antimicrobial activities and represents a promising source of novel antibiotics and secondary metabolites. Among the discovered protein coding genes, 68 % were assigned a putative function, while the remaining 32 % are genes encoding “hypothetical” proteins. Caci_0382 is one of the “hypothetical” proteins that has very few homologs. Sequence analysis shows that the protein belongs to the NTF2-like protein family. The structure of Caci_0382 demonstrates that it shares the same fold and has a similar active site as limonene-1,2-epoxide hydrolase, which suggests that it may have a related function. Using a fluorescence thermal shift assay, we identified stabilizing compounds that suggest potential natural ligands of Caci_0382. Using this information, we determined the crystal structure in complex with trimethylamine to provide a better understanding of the function of this uncharacterized protein.Electronic supplementary materialThe online version of this article (doi:10.1007/s10969-014-9176-z) contains supplementary material, which is available to authorized users.

Saccharin Sulfonamides as Inhibitors of Carbonic Anhydrases I, II, VII, XII, and XIII

A series of modified saccharin sulfonamides have been designed as carbonic anhydrase (CA) inhibitors and synthesized. Their binding to CA isoforms I, II, VII, XII, and XIII was measured by the fluorescent thermal shift assay (FTSA) and isothermal titration calorimetry (ITC). Saccharin bound the CAs weakly, exhibiting the affinities of 1–10 mM for four CAs except CA I where binding could not be detected. Several sulfonamide-bearing saccharines exhibited strong affinities of 1–10 nM towards particular CA isoforms. The functional group binding Gibbs free energy additivity maps are presented which may provide insights into the design of compounds with increased affinity towards selected CAs.

Structure-Thermodynamics Correlations of Fluorinated Benzensulfonamides as Inhibitors of Human Carbonic Anhydrases

The carbonic anhydrases (CA) are established as therapeutic targets [1]. There are 12 catalytically active CA isozymes in human body. At least 30 CA sulfonamide inhibitors have been used as drugs to treat glaucoma, epileptic seizures, altitude sickness, and as diuretics. However, most of them exhibit poor selectivity towards target isozymes and result in various side effects. In this work, a class of 4-substituted-2,3,5,6-tetrafluorobenzensulfonamides as inhibitors of CA is reported. Crystal structures of CAII, CAXII and CAXIII bound with the fluorinated compounds were solved and provided structural details of inhibitor binding. The binding affinity to carbonic anhydrases I, II, VII, XII and XIII was measured by isothermal titration calorimetry and the fluorescent thermal shift assay, and inhibition was determined by stopped-flow CO2 hydration assay. The combined use of these methods has provided a detailed picture of protein-ligand interactions. Experimentally obtained binding data usually depends on various factors including temperature, pH, buffer, etc. In this study, we present intrinsic parameters of binding that are independent of experimental conditions. Structure-thermodynamics correlations were studied using intrinsic parameters. All used biophysical methods have confirmed that fluorinated sulfonamides bound stronger to CA than non-fluorinated, because of the presence of electronegative substituents that decrease the pKa of sulfonamide group and this correlates with an increase in the CA inhibitory properties [2]. A large group of fluorinated compounds possessed nanomolar affinity for selected CAs and several of them were selective towards CAI.

Chemical and Thermal Unfolding of Calreticulin

Calreticulin is a soluble endoplasmic reticulum chaperone, which has a relatively low melting point due to its remarkable structure with a relatively high content of flexible structural elements. Using far ultraviolet circular dichroism (CD) spectroscopy and a fluorescent dye binding thermal shift assay, we have investigated the chemical and thermal stability of calreticulin. When the chemical stability of calreticulin was assessed, a midpoint for calreticulin unfolding was calculated to 3.0M urea using CD data at 222 nm. Using the fluorescent dye binding thermal shift assay, calreticulin was found to obtain a molten structure in urea concentrations between 1-1.5 M urea, and to unfold/aggregate at high and low pH values. The results demonstrated that the fluorescent dye binding assay could measure the thermal stability of calreticulin in aqueous buffers with results comparable to melting points obtained by other techniques.

Characterization of human carbonic anhydrase XII stability and inhibitor binding

Human carbonic anhydrase isozyme XII is a transmembrane protein that is overexpressed in many human cancers. Therefore CA XII is an anticancer drug target. However, there are few compounds that specifically target CA XII. The design of specific inhibitors against CA XII relies on the detailed understanding of the thermodynamics of inhibitor binding and the structural features of the protein–inhibitor complex. To characterize the thermodynamic parameters of the binding of known sulfonamides, namely ethoxzolamide, acetazolamide and trifluoromethanesulfonamide, we used isothermal titration calorimetry and fluorescent thermal shift assay. The binding of these sulfonamides to CA XII was buffer and pH-dependent. Dissection of protonation–deprotonation reactions of both the water molecule bound to the CA XII active site and the sulfonamide group of the inhibitor yielded the intrinsic thermodynamic parameters of binding, such as binding enthalpy, entropy and Gibbs free energy. Thermal shift assay was also used to determine CA XII stabilities at various pH and in the presence of buffers and salts.

Differential Scanning Fluorometry Signatures as Indicators of Enzyme Inhibitor Mode of Action: Case Study of Glutathione S-Transferase

Differential scanning fluorometry (DSF), also referred to as fluorescence thermal shift, is emerging as a convenient method to evaluate the stabilizing effect of small molecules on proteins of interest. However, its use in the mechanism of action studies has received far less attention. Herein, the ability of DSF to report on inhibitor mode of action was evaluated using glutathione S-transferase (GST) as a model enzyme that utilizes two distinct substrates and is known to be subject to a range of inhibition modes. Detailed investigation of the propensity of small molecule inhibitors to protect GST from thermal denaturation revealed that compounds with different inhibition modes displayed distinct thermal shift signatures when tested in the presence or absence of the enzyme's native co-substrate glutathione (GSH). Glutathione-competitive inhibitors produced dose-dependent thermal shift trendlines that converged at high compound concentrations. Inhibitors acting via the formation of glutathione conjugates induced a very pronounced stabilizing effect toward the protein only when GSH was present. Lastly, compounds known to act as noncompetitive inhibitors exhibited parallel concentration-dependent trends. Similar effects were observed with human GST isozymes A1-1 and M1-1. The results illustrate the potential of DSF as a tool to differentiate diverse classes of inhibitors based on simple analysis of co-substrate dependency of protein stabilization.

Abstract 4751: Structure and inhibition of mitogen-activated protein kinase kinase 4 (MEK4): A prostate cancer pro-invasion protein

Abstract Introduction: Metastasis of Prostate Cancer (PCa) represents the second highest cause of death due to cancer among men in the United States. If this unregulated movement of cells can be inhibited, mortality arising from PCa will be greatly reduced. Our group has shown that Mitogen-activated protein kinase kinase 4 (MAP2K4/MEK4), a 399 amino acid protein, activates pro-invasion signaling pathways in human PCa. Further, we have shown that 4,5,7-trihydroxyisoavone (genistein) inhibits MEK4 kinase activity, cell invasion and metastasis of human PCa cells in a murine model. While therapeutically effective, genistein represents a non-chemically optimized natural product with additional undesired effects such as estrogenic receptor stimulation. Recognizing that MEK4 represents a novel and important therapeutic target, we have sought to characterize its structure, biochemical function and have synthesized genistein analogs designed to target it. Summary of Results: Using X-ray crystallography we have obtained a 3.38Å resolution structure of the MEK4 catalytic domain. We are in the process of improving our resolution, and of deriving 3D structures with inhibitors bound to MEK4. We have evaluated biophysical interactions between MEK4 and ligands (i.e., genistein and its analogs). Ligand binding to a protein tends to increase its stability, thereby increasing the temperature of denaturation, or melting. Using a fluorescence thermal shift (FTS) assay, we demonstrated that genistein and 6 of 39 novel analogs thermally stabilized MEK4 in a concentration dependent manner, consistent with MEK4 binding. We identified a similar pattern in 5 compounds from a commercial library, one of which was staurosporine - a broad spectrum protein kinase inhibitor. We developed and characterized a novel in vitro MEK4 kinase assay, using kinase dead p38MAPK (K53A) as substrate. In human PCa cells, MEK4 activates the p38 MAPK pro-invasion pathway. To date, we have used this assay system to demonstrate that staurosporine inhibits MEK4 with nanomolar IC50 values. Conclusions and Future Directions: MEK4 is a novel target for therapeutic intervention in PCa. Our ultimate goal is to synergistically utilize structural and biochemical information to inform the synthesis of inhibitors that specifically and selectively act upon MEK4 to inhibit human PCa metastasis in man. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4751. doi:1538-7445.AM2012-4751

Environment sensing and response mediated by ABC transporters

BackgroundTransporter proteins are one of an organism’s primary interfaces with the environment. The expressed set of transporters mediates cellular metabolic capabilities and influences signal transduction pathways and regulatory networks. The functional annotation of most transporters is currently limited to general classification into families. The development of capabilities to map ligands with specific transporters would improve our knowledge of the function of these proteins, improve the annotation of related genomes, and facilitate predictions for their role in cellular responses to environmental changes.ResultsTo improve the utility of the functional annotation for ABC transporters, we expressed and purified the set of solute binding proteins from Rhodopseudomonas palustris and characterized their ligand-binding specificity. Our approach utilized ligand libraries consisting of environmental and cellular metabolic compounds, and fluorescence thermal shift based high throughput ligand binding screens. This process resulted in the identification of specific binding ligands for approximately 64% of the purified and screened proteins. The collection of binding ligands is representative of common functionalities associated with many bacterial organisms as well as specific capabilities linked to the ecological niche occupied by R. palustris.ConclusionThe functional screen identified specific ligands that bound to ABC transporter periplasmic binding subunits from R. palustris. These assignments provide unique insight for the metabolic capabilities of this organism and are consistent with the ecological niche of strain isolation. This functional insight can be used to improve the annotation of related organisms and provides a route to evaluate the evolution of this important and diverse group of transporter proteins.

Thermal Stability Assay for Glaucoma‐Causing Myocilin Mutants using Maltose Binding Fusion Protein (MBP)

Myocilin is a protein closely linked to inherited forms of glaucoma, a neurodegenerative disorder associated with increased intraocular pressure. In myocilin, over 90% of reported glaucoma‐causing missense mutations are localized to the C‐terminal olfactomedin (OLF) domain. To determine the relative stability of wild‐type and mutant OLF, we developed a fluorescence thermal stability assay without removal of MBP. The fluorescence thermal shift assay can accommodate relatively low concentrations of protein (0.25–0.5 mg/ml) and uses a real time PCR (RT‐PCR) instrument to conduct a slow melt while measuring fluorescence. The excitation and emission settings of the RT‐PCR instrument are compatible with Sypro Orange, a dye known to bind to hydrophobic regions of proteins, which become increasingly exposed as the temperature of the protein‐dye solution is raised. Given upper and lower limits of the fluorescence signal, a melting point can be determined as the midpoint of the unfolding transition. We expressed and purified the OLF (amino acids 228–504) domain of myocilin and select glaucoma mutants (D380A, I477N, I477S, K423E) as maltose‐binding fusion (MBP) proteins in E. coli. In comparison to wild‐type, all four MBP‐OLF mutants tested were destabilized but retained a discrete melting transition expected for a folded protein. Our study appears to be a novel application of the fluorescence stability assay to a MBP fusion protein and lays the foundation for the identification of tailored therapeutic molecules to delay the onset of glaucoma. This work was supported by NSF REU #0851780, Glaucoma Research Foundation (Shaffer Fund), and American Health Assistance Foundation.