High-Throughput Fragment Screening by Affinity LC-MS

Abstract

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.

Immobilized lipodisks as model membranes in high-throughput HPLC-MS analysis

“…It should be noted that the maximum number of analytes in each mixture likely is considerable higher than seven, as was used in the present study to avoid overlapping massto-charge ratios. Duong-Thi and coauthors have recently verified high throughput analysis in a similar, but lipodisk-free, system using up to 65 compounds in each mixture (11). The robust chromatographic medium, together with the possibility to analyze multiple compounds simultaneously, makes the current lipodisk-HPLC-MS system far more efficient for drug partition studies than previous systems based on immobilization of lipodisks to gel filtration media (16,17).…”

Weak Affinity Chromatography for Evaluation of Stereoisomers in Early Drug Discovery

“…Another benefit with this technique is that a high throughput of thousands of fragments per day can be achieved. 6 The present work demonstrates the applicability of WAC with MS detection to determine the affinity of stereoisomeric fragments to thrombin, an important serine protease in the coagulation cascade. Screening was performed with enantiomeric as well as diastereomeric mixtures of fragments and elaborated fragments, respectively.…”

“…Retention (t spec ) of three reference binders (0.1 mM BZA, 3-ABA, and 4-ABA) was used to determine B tot of thrombin columns 1 and 2. It was done by comparing the average retention (t spec ) of the reference compounds with previously obtained values, under identical conditions, using thrombin columns with a known B tot (determined with frontal chromatography 6 ). Assuming that equation (1) …”

“…; Agilent Technologies, Waldbronn, Germany). The immobilization procedure, which has been described in detail previously, 6,8 included oxidation of diol silica to aldehyde silica followed by covalent coupling to primary amino groups of thrombin by reductive amination chemistry (Schiff base method). The reaction creates covalent bonds (aminolinks) between the high-performance silica support and the protein.…”

“…9 Two reference columns, with the same size and silica material as the active ones, were used to estimate nonspecific binding. The first of these reference columns contained PPACKinhibited thrombin (inhibition procedure has been reported previously 6 ), and it was used to quantify binding to noncatalytic sites of the protein. The second reference column was a blank diol-silica column that was used to determine binding to the supporting silica material.…”