Anti-adhesion drugs may be an alternative to antibiotics to control infection of micro-organisms. The well-characterized interaction between cholera toxin and the cellular glycolipid GM1 makes it an attractive model for inhibition studies in general. In this report, we demonstrate a high-performance liquid affinity chromatography approach called weak affinity chromatography to evaluate cholera toxin inhibitors. The cholera toxin B-subunit was covalently coupled to porous silica and a (weak) affinity column was produced. The K(D) values of galactose and meta-nitrophenyl alpha-d-galactoside were determined with weak affinity chromatography to be 52 and 1 mm, respectively, which agree well with IC(50) values previously reported. To increase inhibition potency multivalent inhibitors have been developed and the interaction with multivalent glycopolypeptides was also evaluated. The affinity of these compounds was found to correlate with the galactoside content but K(D) values were not obtained because of the inhomogeneous response and slow off-rate from multivalent interactions. Despite the limitations in obtaining direct K(D) values of the multivalent galactopolypeptides, weak affinity chromatography represents an additional and valuable tool in the evaluation of monovalent as well as multivalent cholera toxin inhibitors. It offers multiple advantages, such as a low sample consumption, high reproducibility and short analysis time, which are often not observed in other methods of analysis.
Aberrant glycosylation is connected to several pathological conditions and lectins are useful tools to characterize glycosylated biomarkers. The Aleuria aurantia lectin (AAL) is of special interest since it interacts with all types of fucosylated saccharides. AAL has been expressed in E.coli as a fully functional recombinant protein. Engineered variants of AAL have been developed with the aim of creating monovalent lectins with more homogenous binding characteristics. Four different forms of AAL were studied in the present work: native AAL purified from Aleuria aurantia mushrooms, recombinant AAL dimer, recombinant AAL monomer and recombinant AAL site 2 (S2-AAL). The affinities of these AAL forms towards a number of saccharides were determined with weak affinity chromatography (WAC). Disaccharides with fucose linked α1-3 to GlcNAc interacted with higher affinity compared to fucose linked α1-6 or α1-4 and the obtained dissociation constants (Kd) were in the range of 10 μM for all AAL forms. Tetra- and pentasaccharides with fucose in α1-2, α1-3 or α1-4 had Kd values ranging from 0.1–7 mM while a large α1-6 fucosylated oligosaccharide had a Kd of about 20 μM. The recombinant multivalent AAL forms and native AAL exhibited similar affinities towards all saccharides, but S2-AAL had a lower affinity especially regarding a sialic acid containing fucosylated saccharide. It was demonstrated that WAC is a valuable technique in determining the detailed binding profile of the lectins. Specific advantages with WAC include a low consumption of non-labeled saccharides, possibility to analyze mixtures and a simple procedure using standard HPLC equipment.
An immunoadsorbent based on immobilized C1q has been developed to remove possibly pathogenic immune complexes from plasma deriving from patients suffering from systemic lupus erythematosus or rheumatoid arthritis. Traditional immobilization procedures based on, e.g., cyanogen bromide activation could not be used to produce an efficient adsorbent. However, by using antibodies directed towards C1q as handles for the immobilization of C1q it was possible to make an adsorbent that efficiently bound immune complexes in plasma. The capacity of the C1q anti-C1q adsorbent to bind artificial immune complexes such as aggregated human globulins or immune complexes from various plasma samples was evaluated. Both batch and column experiments were conducted. The typical capacity in batch was about 1 mg immune complexes/ml gel when incubated with patient plasma samples with high titers of immune complexes. Special attention has to be paid to leakage of undesirable components from the adsorbent. It was found that leakage of C1q occurred but it was not more than after covalent immobilization procedures such as cyanogen bromide.
In this study, we compared affinity data from surface plasmon resonance (SPR) and weak affinity chromatography (WAC), two established techniques for determination of weak affinity (mM-mu M) small molecule-protein interactions. In the current comparison, thrombin was used as target protein. In WAC the affinity constant (K-D) was determined from retention times, and in SPR it was determined by Langmuir isotherm fitting of steady-state responses. Results indicate a strong correlation between the two methods (R-2 = 0.995, P < 0.0001). (C) 2014 Elsevier Inc. All rights reserved.
Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures.
Fragment screening, an emerging approach for hit finding in drug discovery, has recently been proven effective by its first approved drug, vemurafenib, for cancer treatment. Techniques such as nuclear magnetic resonance, surface plasmon resonance, and isothemal titration calorimetry, with their own pros and cons, have been employed for screening fragment libraries. As an alternative approach, screening based on high-performance liquid chromatography separation has been developed. In this work, we present weak affinity LC/MS as a method to screen fragments under high-throughput conditions. Affinity-based capillary columns with immobilized thrombin were used to screen a collection of 590 compounds from a fragment library. The collection was divided into 11 mixtures (each containing 35 to 65 fragments) and screened by MS detection. The primary screening was performed in < 4 h (corresponding to > 3500 fragments per day). Thirty hits were defined, which subsequently entered a secondary screening using an active site-blocked thrombin column for confirmation of specificity. One hit showed selective binding to thrombin with an estimated dissociation constant (K-D) in the 0.1 mM range. This study shows that affinity LC/MS is characterized by high throughput, ease of operation, and low consumption of target and fragments, and therefore it promises to be a valuable method for fragment screening.
In early drug discovery (e.g. in fragment screening), recognition of stereoisomeric structures is valuable and guides medicinal chemists to focus only on useful configurations. In this work, we concurrently screened mixtures of stereoisomers and estimated their affinities to a protein target (thrombin) using weak affinity chromatography-mass spectrometry (WAC-MS). Affinity determinations by WAC showed that minor changes in stereoisomeric configuration could have major impact on affinity. The ability of WAC-MS to provide instant information about stereoselectivity and binding affinities directly from analyte mixtures is a great advantage in fragment library screening and drug lead development.
Fragment-based drug design (FBDD) is currently being implemented in drug discovery, creating a demand for developing efficient techniques for fragment screening. Due to the intrinsic weak or transient binding of fragments (mM–uM in dissociation constant (KD)) to targets, methods must be sensitive enough to accurately detect and quantify an interaction. This study presents weak affinity chromatography (WAC) as an alternative tool for screening of small fragments. The technology was demonstrated by screening of a selected 23 compound fragment collection of documented binders, mostly amidines, using trypsin and thrombin as model target protease proteins. WAC was proven to be a sensitive, robust, and reproducible technique that also provides information about affinity of a fragment in the range of 1 mM–10uM. Furthermore, it has potential for high throughput as was evidenced by analyzing mixtures in the range of 10 substances by WAC–MS. The accessibility and flexibility of the technology were shown as fragment screening can be performed on standard HPLC equipment. The technology can further be miniaturized and adapted to the requirements of affinity ranges of the fragment library. All these features of WAC make it a potential method in drug discovery for fragment screening.
In this report we have evaluated the potential of using fluorescence/Förster resonance energy transfer (FRET) in a competitive immunosensor for continuous monitoring of the carbohydrate hapten maltose. The cyanine dyes Cy5 and Cy5.5 were used as a donor–acceptor pair by conjugation to maltose-labeled bovine serum albumin (BSA) and the monoclonal antibody IgG 39.5, giving Cy5–BSA–maltotriitol (3.1/1/18) and Cy5.5–mAb39.5 (2.2/1), respectively. This antibody with weak affinity towards maltose showed full reversibility to both the free maltose and the maltose-labeled conjugate. It allowed us to measure continuously the maltose content by monitoring the FRET signal change over time due to displacement of Cy5–BSA–maltotriitol from Cy5.5–mAb39.5 inside a semipermeable capsule. A near 22% total increase was seen in the fluorescence intensity ratio I670/I700 in the presence of maltose, with a calculated EC50 = 1.87 ± 0.13 mM (R2 = 0.9984) from the sigmoidal dose–response curve at 25 °C. Specificity of the immunosensor was shown with the structural analog to maltose, cellobiose, and it generated no detectable response. A minor drift in the sensor baseline was seen with 0.4% per 24 h, which was in the same magnitude as the signal-to-noise ratio, during the 4 weeks of measurements. The immunosensor was applied to crude samples of oat drinks for direct quantification of the maltose content. Overall, this work demonstrates the potential to use an immunosensor based on weakly binding antibodies and FRET technology for remote and non-invasive carbohydrate monitoring.
Continuous monitoring of drug levels and endogenous molecules in biological fluids is a developing research area with many applications. One example is the need to improve life for millions of diabetes mellitus patients by continuously monitoring the glucose level. In order to have a dynamic response, the recognition molecule in a continuous sensor should preferentially have a fast dissociation rate and a dissociation constant in the millimolar range. We have evaluated the monoclonal antibody (mAb) 3F1E8-A2 for its potential to be used in a future glucose sensor application. The mAb was generated from hybridomas by immunizing mice with 10 kDa dextran (an α1,6-glucose polymer) with the aim of obtaining mAbs that can recognize the glucose monomer. The mAb was immobilized to macroporous silica and the interaction with dextran-derived oligosaccharides was evaluated with weak affinity chromatography (WAC). To measure the low affinities between the mAb 3F1E8-A2 and different monosaccharides, a competitive weak affinity chromatography approach was employed. It was found that the mAb had a higher specificity for glucose compared with other monosaccharides and the dissociation constant (Kd) towards glucose was determined as 18.8 ± 2.6 mm.
By examining the interactions between the protein hen egg-white lysozyme (HEWL) and commercially available and chemically synthesized carbohydrate ligands using a combination of weak affinity chromatography (WAC), NMR spectroscopy and molecular simulations, we report on new affinity data as well as a detailed binding model for the HEWL protein. The equilibrium dissociation constants of the ligands were obtained by WAC but also by NMR spectroscopy, which agreed well. The structures of two HEWL-disaccharide complexes in solution were deduced by NMR spectroscopy using (1)H saturation transfer difference (STD) effects and transferred (1)H,(1)H-NOESY experiments, relaxation-matrix calculations, molecular docking and molecular dynamics simulations. In solution the two disaccharides β-d-Galp-(1→4)-β-D-GlcpNAc-OMe and β-D-GlcpNAc-(1→4)-β-D-GlcpNAc-OMe bind to the B and C sites of HEWL in a syn-conformation at the glycosidic linkage between the two sugar residues. Intermolecular hydrogen bonding and CH/π-interactions form the basis of the protein-ligand complexes in a way characteristic of carbohydrate-protein interactions. Molecular dynamics simulations with explicit water molecules of both the apo-form of the protein and a ligand-protein complex showed structural change compared to a crystal structure of the protein. The flexibility of HEWL as indicated by a residue-based root-mean-square deviation analysis indicated similarities overall, with some residue specific differences, inter alia, for Arg61 that is situated prior to a flexible loop. The Arg61 flexibility was notably larger in the ligand-complexed form of HEWL. N,N'-Diacetylchitobiose has previously been observed to bind to HEWL at the B and C sites in water solution based on (1)H NMR chemical shift changes in the protein whereas the disaccharide binds at either the B and C sites or the C and D sites in different crystal complexes. The present study thus highlights that protein-ligand complexes may vary notably between the solution and solid states, underscoring the importance of targeting the pertinent binding site(s) for inhibition of protein activity and the advantages of combining different techniques in a screening process.
The marked increase in demand for contraceptives and anti-inflammatory agents such as cortisol and prednisolone, combined with a diminishing supply of steroid raw materials may lead to shortages of steroid drugs1. Thus it is important to develop new sources for steroids as well as to devise more efficient means for steroid conversions. Here we report a new approach to steroid transformation in which activated immobilised microorganisms are utilised and which represents a promising alternative to conventional microbial transformation processes.
Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K (D)) = mM -aEuro parts per thousand mu M) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K (D) < 200 mu M were detected, including a few highly efficient GAK binders (K (D) of 2 mu M; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.
Lipodisks, also referred to as polyethylene glycol (PEG)-stabilized bilayer disks, have previously been demonstrated to hold great potential as model membranes in drug partition studies. In this study, an HPLC-MS system with stably immobilized lipodisks is presented. Functionalized lipodisks were immobilized on two different HPLC support materials either covalently by reductive amination or by streptavidin-biotin binding. An analytical HPLC column with immobilized lipodisks was evaluated by analysis of mixtures containing 15 different drug compounds. The efficiency, reproducibility, and stability of the system were found to be excellent. In situ incorporation of cyclooxygenase-1 (COX-1) in immobilized lipodisks on a column was also achieved. Specific binding of COX-1 to the immobilized lipodisks was validated by interaction studies with QCM-D. These results, taken together, open up the possibility of studying ligand interactions with membrane proteins by weak affinity chromatography.
The increasing use of fragment-based lead discovery (FBLD) in industry as well as in academia creates a high demand for sensitive and reliable methods to detect the binding of fragments to act as starting points in drug discovery programs. Nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and X-ray crystallography are well-established methods for fragment finding, and thermal shift and fluorescence polarization (FP) assays are used to a lesser extent. Weak affinity chromatography (WAC) was recently introduced as a new technology for fragment screening. The study presented here compares screening of 111 fragments against the ATPase domain of HSP90 by all of these methods, with isothermal titration calorimetry (ITC) used to confirm the most potent hits. The study demonstrates that WAC is comparable to the established methods of ligand-based NMR and SPR as a hit-id method, with hit correlations of 88% and 83%, respectively. The stability of HSP90 WAC columns was also evaluated and found to give 90% reproducibility even after 207 days of storage. A good correlation was obtained between the various technologies, validating WAC as an effective technology for fragment screening.