Enzyme kinetics by GH7 cellobiohydrolases on chromogenic substrates is dictated by non-productive binding: insights from crystal structures and MD simulation.

Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) (EC3.2.1.176) are the major cellulose degrading enzymes both in industrial settings and in the context of carbon cycling in nature. Small carbohydrate conjugates such as p-nitrophenyl-ß-d-cellobioside (pNPC), p-nitrophenyl-ß-d-lactoside (pNPL) and methylumbelliferyl-ß-d-cellobioside have commonly been used in colorimetric and fluorometric assays for analysing activity of these enzymes. Despite the similar nature of these compounds the kinetics of their enzymatic hydrolysis vary greatly between the different compounds as well as among different enzymes within the GH7 family. Through enzyme kinetics, crystallographic structure determination, molecular dynamics simulations, and fluorometric binding studies using the closely related compound o-nitrophenyl-ß-d-cellobioside (oNPC), in this work we examine the different hydrolysis characteristics of these compounds on two model enzymes of this class, TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium. Protein crystal structures of the E212Q mutant of TrCel7A with pNPC and pNPL, and the wildtype TrCel7A with oNPC, reveal that non-productive binding at the product site is the dominating binding mode for these compounds. Enzyme kinetics results suggest the strength of non-productive binding is a key determinant for the activity characteristics on these substrates, with PcCel7D consistently showing higher turnover rates (kcat ) than TrCel7A, but higher Michaelis-Menten (KM ) constants as well. Furthermore, oNPC turned out to be useful as an active-site probe for fluorometric determination of the dissociation constant for cellobiose on TrCel7A but could not be utilized for the same purpose on PcCel7D, likely due to strong binding to an unknown site outside the active site.

Weak affinity chromatography as a new approach for fragment screening in drug discovery.

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-µM in dissociation constant (K(D))) 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-10 µM. 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.

Toward high-throughput drug screening on a chip-based parallel affinity separation platform.

High-throughput screening of compound libraries, including the study of fragments, has become one of the cornerstones in modern drug discovery research. During this process hits are defined that may be developed into valuable leads and eventually into possible drug candidates. In this paper, we have demonstrated that parallel zonal weak affinity chromatography in microcolumns on a chip offers a possible screening format for weakly binding ligands toward a protein target. We used albumin as a model system because this transport protein is well established as a binder (both weak and strong) for drug substances. Bovine serum albumin was immobilized on microparticulate diolsilica particles and then packed into a 24-channel cartridge, which served as the separation platform. Analysis of the obtained chromatograms yielded information about affinity even in the millimolar range. Employing this approach, thousands of substances can be screened in just a day. We feel confident that zonal affinity chromatography will provide a useful technology in the future for performing high-throughput screening.

pH dependency of ligand binding to cellobiohydrolase 1 (Cel7A). Affinity, selectivity and inhibition for designed propranolol analogues.

The affinity and enantioselectivity have been determined for designed propranolol derivatives as ligands for Cel7A by capillary electrophoresis (CE) at pH 7.0. These results have been compared to measurements at pH 5.0. In agreement with previous studies, the affinity increased at the higher pH. However, the affinity was not as dependent of the ligand structure at pH 7.0 as at pH 5.0, and the selectivity was generally decreased. Instead, at pH 7.0, the changes in binding were mainly dependent on the presence of additional dihydroxyl groups, indicating an increased importance of the electrostatic interactions. To evaluate the pH dependent variations in binding, changes in both the ligand and in the enzyme had to be taken into account. To ensure that the ligands had the same charge in all measurements, pKa-values of all compounds were determined. The ligand-protein interaction has also been studied by inhibition experiments at both pHs to evaluate the specific binding to the active site when competing with the substrate p-nitrophenyl lactoside (pNPL). With support of docking computations we propose a hypothesis on the effect of the ligand structure and pH dependency of the binding and selectivity of amino alcohols to Cel7A.

Migration time and peak area artifacts caused by systemic effects in voltage controlled capillary electrophoresis.

The effect of an artificial conductivity step on the migration time for rac-propranolol was investigated with the purpose to give a strict physical background to the intensively studied migration time and integration variations in voltage stabilized capillary electrophoresis. The experiments verified a theoretical derivation of migration time disturbances caused by the sometimes unavoidable conductivity variations in the system. The verified equation describing migration time variations was used for simulations of a number of relevant cases, such as variation in injected sample conductivity, post-injection diffusional broadening of the sample zone, and the physical effect of a selector zone in the partial filling mode. A similar theoretical and experimental treatment of the integration errors caused by non-uniform velocity was also included with special reference to enantiomer separation in the partial filling mode. The same experiments were also used to show that the standard migration time related method for integration correction fails if the velocity of a component is not constant throughout the experiment, such as the situation when a selector is employed in partial-filling mode.

Lectin affinity capillary electrophoresis in glycoform analysis applying the partial filling technique.

The study of protein glycosylation and its significance in biological interactions is a field of growing interest. This work demonstrates a lectin-based separation of protein glycoforms of alpha1-acid glycoprotein (AGP or orosomucoid) with capillary electrophoresis. Glycoform analysis was performed with a "partial filling technique" with the lectin Concanavalin A (Con A) as affinity ligand. Con A separated human AGP into two peaks; the first peak included AGP glycoforms without biantennary glycans, and the second peak represented the fraction that had one or more biantennary glycans. The applicability of the method was demonstrated with the analysis of AGP from clinical samples and AGP treated with N-glycosidase F. The AGP separation was also used as a reporter system to estimate the dissociation constant (KD) between Con A and a competing sugar.

Weak affinity chromatography for evaluation of stereoisomers in early drug discovery.

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 a 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.

High-throughput fragment screening by affinity LC-MS.

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.

Determination of dissociation constants between polyelectrolytes and proteins by affinity capillary electrophoresis.

In this manuscript we report the binding affinity between two model proteins, human serum albumin (HSA) and ribonuclease A (RNase A), and negatively charged polyelectrolytes, two different heparin fractions and dextran sulfate, by means of partial filling and affinity capillary electrophoresis. The apparent dissociation constants, K(d), obtained by use of the partial-filling method, between HSA and heparin (17kDa), heparin (3kDa) and dextran sulfate (8kDa) were 33 and 307microM, respectively. A new method was developed to determine affinities that take in account different migration directions between the protein and the polyelectrolyte, which was required to study RNase A. By use of this affinity capillary electrophoresis two K(d) values were observed for the interaction between RNase A and heparin 17kDa, yielding a high affinity binding with K(d1) 0.0075microM, and a lower affinity binding with K(d2) 8.7microM. For dextran sulfate 8kDa these K(d) values were 0.027 and 10.4microM, respectively. Heparin 3kDa only showed a single K(d) value of 0.52microM. The results show that the magnitude of the binding affinity depends on the type of polyelectrolyte and its molecular weight.

Compounds binding to the S2-S3 pockets of thrombin.

A set of compounds designed to bind to the S2-S3 pockets of thrombin was prepared. These compounds included examples with no interactions in the S1 pocket. Proline, a common P2 in many thrombin inhibitors, was combined with known P3 residues and P1 substituents of varying size and lipophilicity. Binding constants were determined using surface plasmon resonance (SPR) biosensor technology and were found to be in good agreement with results from an enzyme assay. A dramatic increase in affinity (100-1000 times) was seen for compounds incorporating an amino group capable of forming a hydrogen bond with gly216 in the protein backbone. The ligand efficiency was increased by including substituents that form stronger hydrophobic interactions with the P1 pocket. The binding mode was confirmed by X-ray analysis, which revealed the anticipated binding motif that included hydrogen bonds as well as a tightly bound water molecule. A QSAR model indicated that hydrogen bonding and lipophilicity were important for the prediction of binding constants. The results described here may have implications for how directed compound libraries for shallow protein pockets, like S2 and S3 in serine proteases, can be designed.

Thermodynamical studies of designed ligands binding to Cel7A using partial-filling capillary electrophoresis.

A convenient experimental method for thermodynamical studies based on partial-filling affinity CE is presented. The advantages of this approach are the possibility to determine binding energies from relatively weak interactions as well as the small amounts of samples consumed. In order to explore the affinity and selectivity of the cellobiohydrolase Cel7A, a number of propranolol analogues were recently designed. The affinities of a selection of these ligands were determined in the temperature interval 15-40 degrees C, and DeltaG degrees , DeltaH degrees and DeltaS degrees were obtained by means of Van't Hoff plots. Through these experiments, the importance of the entropy contribution in the complexation between the ligands and Cel7A has been demonstrated.

Bilayer disk capillary electrophoresis: a novel method to study drug partitioning into membranes.

CE in the presence of lipid bilayer disks was introduced as a new approach in membrane partitioning studies. The disks were used as a pseudostationary phase in the partial-filling mode of CE and the partitioning of cationic drugs was determined. The migration times of the analytes increased linearly with the lipid amount in the system. An appropriate algorithm for the calculation of a partition coefficient is presented. In the disk-shaped bilayers, which have excellent stability and shelf life, all of the lipids are readily available for interaction and the disks can be used as realistic cell membrane models.

New propranolol analogues: binding and chiral discrimination by cellobiohydrolase Cel7A.

Novel propranolol analogues have been designed and synthesised and their enantioselective binding to the cellulose degrading enzyme, Cel7A, has been evaluated. Affinity and enantioselectivity have been determined by capillary electrophoresis experiments. Ligands with significantly improved affinity and selectivity have been obtained and an analysis of the results has led to insights concerning the relation between the changes in ligand structure and selectivity as well as affinity to the protein.

Screening for transient biological interactions as applied to albumin ligands: a new concept for drug discovery.

The notion that many biological interactions are based on transient binding (dissociation constants (K(d)) in the range of 10-0.01 mM) is familiar, yet the implications for biological sciences have been realized only recently. An important area of biological sciences is drug design, where the traditional "lock and key" view of binding has prevailed and drug candidates are usually selected on their merits as being tight binders. However, the rationale that transient interactions are of importance for drug discovery is slowly gaining acceptance. These interactions may relate not only to the desired target interaction but also to unwanted interactions creating, for example, toxicity problems. Here we demonstrate, in a high-throughput screening format, affinity selection of weak binders to a model target of albumin by zonal retardation chromatography. It is perceived that this approach can define the "transient drug" as a complement to current drug discovery procedures.

Determination of dissociation constants by competitive binding in partial filling capillary electrophoresis.

The determination of dissociation constands (K(d)) by competitive ligand binding in partial filling capillary electrophoresis is demonstrated. Two different strategies were applied, one of which only uses a single reporter ligand and a more elaborated one which suppresses systemic disturbances by using a racemic mixture as reporter. The dissociation constants obtained by both alternatives were virtually identical and in good agreement with those previously reported.

Determination of protein-ligand affinity constants from direct migration time in capillary electrophoresis.

A simple method to calculate dissociation constants for protein-ligand interactions by partial-filling capillary electrophoresis is demonstrated. The method uses raw migration time data for the ligand and needs only additional information about capillary inner radius and the absolute amount of protein loaded. A theoretical study supported by experimental data also demonstrates that the retention of analyte in affinity capillary electrophoresis (ACE) using the partial-filling technique depends linearly on the absolute amount of selector added but is independent of both selector zone length and selector mobility. Factors such as field strength and electroosmotic flow are also cancelled out if they are kept constant. The theory is confirmed and the usefulness of the method is demonstrated by enantioseparations using alpha-acid glycoprotein (AGP) and cellulase (Cel 7A) as chiral selectors.

A statistical experimental design to study factors affecting enantioseparation of propranolol by capillary electrophoresis with cellobiohydrolase (Cel7A) as chiral selector.

The capillary electrophoretic enantioseparation of rac-propranolol using cellobiohydrolase Tr Cel7A as selector was optimized by an unbiased statistical experimental design. A set of pre-experiments was performed in order to identify critical experimental factors. In the definitive chemometric design pH, ranging from 5 to 7, ionic strength ranging between 0.01 and 0.02 and organic solvent additive in concentration from 1 to 19% v/v were studied. The response surface plot revealed a separation optimum in the pH interval studied. When all parameters were taken into account, a background electrolyte consisting of 0.016 M bistris-acetate buffer with pH 6.5 and 17% v/v acetonitrile gave the optimum separation. The significance of the statistical design was confirmed by the generally good agreement obtained between predicted response and actual experimental data.