Gerdien E. de Kloe

Transforming fragments into candidates: small becomes big in medicinal chemistry

Fragment-based drug discovery (FBDD) represents a logical and efficient approach to lead discovery and optimisation. It can draw on structural, biophysical and biochemical data, incorporating a wide range of inputs, from precise mode-of-binding information on specific fragments to wider ranging pharmacophoric screening surveys using traditional HTS approaches. It is truly an enabling technology for the imaginative medicinal chemist. In this review, we analyse a representative set of 23 published FBDD studies that describe how low molecular weight fragments are being identified and efficiently transformed into higher molecular weight drug candidates. FBDD is now becoming warmly endorsed by industry as well as academia and the focus on small interacting molecules is making a big scientific impact.

Surface Plasmon Resonance Biosensor Based Fragment Screening Using Acetylcholine Binding Protein Identifies Ligand Efficiency Hot Spots (LE Hot Spots) by Deconstruction of Nicotinic Acetylcholine Receptor α7 Ligands

The soluble acetylcholine binding protein (AChBP) is a homologue of the ligand-binding domain of the nicotinic acetylcholine receptors (nAChR). To guide future fragment-screening using surface plasmon resonance (SPR) biosensor technology as a label-free, direct binding, biophysical screening assay, a focused fragment library was generated based on deconstruction of a set of α7 nAChR selective quinuclidine containing ligands with nanomolar affinities. The interaction characteristics of the fragments and the parent compounds with AChBP were evaluated using an SPR biosensor assay. The data obtained from this direct binding assay correlated well with data from the reference radioligand displacement assay. Ligand efficiencies for different (structural) groups of fragments in the library were correlated to binding with distinct regions of the binding pocket, thereby identifying ligand efficiency hot spots (LE hot spots). These hot spots can be used to identity the most promising hit fragments in a large scale fragment library screen.

Online Fluorescence Enhancement Assay for the Acetylcholine Binding Protein with Parallel Mass Spectrometric Identification

The acetylcholine binding protein (AChBP) is considered an analogue for the ligand-binding domain of neuronal nicotinic acetylcholine receptors (nAChRs). Its stability and solubility in aqueous buffer allowed the development of an online bioaffinity analysis system. For this, a tracer ligand which displays enhanced fluorescence in the binding pocket of AChBP was identified from a concise series of synthetic benzylidene anabaseines. Evaluation and optimization of the bioaffinity assay was performed in a convenient microplate reader format and subsequently transferred to the online format. The high reproducibility has the prospect of estimating the affinities of ligands from an in-house drug discovery library injected in one known concentration. Furthermore, the online bioaffinity analysis system could also be applied to mixture analysis by using gradient HPLC. This led to the possibility of affinity ranking of ligands in mixtures with parallel high-resolution mass spectrometry for compound identification.

Fragment library screening reveals remarkable similarities between the G protein-coupled receptor histamine H4 and the ion channel serotonin 5-HT3A

Graphical abstract

Small and colorful stones make beautiful mosaics: Fragment-Based Chemogenomics

Smaller stones with a wide variety of colors make a higher resolution mosaic. In much the same way, smaller chemical entities that are structurally diverse are better able to interrogate protein binding sites. This feature article describes the construction of a diverse fragment library and an analysis of the screening of six representative protein targets belonging to three diverse target classes (G protein-coupled receptors ADRB2, H1R, H3R, and H4R, the ligand-gated ion channel 5-HT3R, and the kinase PKA) using chemogenomics approaches. The integration of experimentally determined bioaffinity profiles across related and unrelated protein targets and chemogenomics analysis of fragment binding and protein structure allow the identification of: (i) unexpected similarities and differences in ligand binding properties, and (ii) subtle ligand affinity and selectivity cliffs. With a wealth of fragment screening data being generated in industry and academia, such approaches will contribute to a more detailed structural understanding of ligand-protein interactions.

Fragment library design: efficiently hunting drugs in chemical space

With the growth in fragment-based drug discovery, numerous strategies have been described for the design of fragment libraries. Key choices need to be made on both the selection criteria to be applied and the source of the fragments in the library. Here we review some of the key trends and recent developments in the rapidly evolving field of fragment library design, providing an overview of current design strategies and surveying the characteristics of published fragment libraries.

Interaction Kinetic and Structural Dynamic Analysis of Ligand Binding to Acetylcholine-Binding Protein

The mechanism of agonist interactions with Cys-loop ligand-gated ion channels has been studied using the acetylcholine-binding protein (AChBP) from Lymnaea stagnalis as a model protein and acetylcholine, nicotine, epibatidine, and a series of substituted quinuclidines as ligands. A biosensor-based assay for direct interaction studies of immobilized AChBP and small molecule ligands was developed. It allowed the characterization of the interaction kinetics of the ligands and the structural dynamics of the protein. The interactions with AChBP were very sensitive to variations in the experimental conditions and showed several types of complexities. These could be resolved into two types of ligand-induced secondary effects with different kinetics, representing fast and slow conformational changes. The data could be rationalized in a mechanistic model, and a structural interpretation of the interaction was obtained by molecular modeling involving induced fit and loop flexibility simulations. The data suggest that AChBP exhibits ligand-induced structural dynamics, as expected for the ligand gating mechanism of Cys-loop receptors. It shows that the formation of the initial encounter complex between AChBP and ligands is very rapid, in accordance with the functional characteristics required of neurotransmission. These developed procedures will enable further exploration of the mechanism of Cys-loop receptor function and the identification of specific ligands suitable for pharmacological use.

High-Resolution Bioactivity Profiling of Mixtures toward the Acetylcholine Binding Protein Using a Nanofractionation Spotter Technology

This study describes the evaluation, validation, and use of contactless postcolumn fractionation of bioactive mixtures with acetylcholine binding protein (AChBP) affinity analysis with help of a spotter technology. The high-resolution fractionation tailors the fractionation frequency to the chromatographic peaks. Postcolumn reagents for AChBP bioaffinity profiling are mixed prior to droplet ejection into 1536-well plates. After an incubation step, microplate reader analysis is used to determine bioactive compounds in a mixture. For ligands tested, a good correlation was found for IC50s determined in flow injection analysis mode when compared with traditional radioligand binding assays. After the evaluation and validation, bioaffinity profiling of actual mixtures was performed. The advantage of this “atline” technology using postcolumn bioaffinity analysis when compared to continuous flow online postcolumn bioaffinity profiling is the possibility to choose postcolumn incubation times freely without compromising resolution due to diffusion effects.

Online parallel fragment screening and rapid hit exploration for nicotinic acetylcholine receptors

An online bioaffinity analysis system was used to screen our in-house fragment library on two related proteins, Ls- and Ac-AChBP, model proteins for nAChRs, in particular the α7 subtype. An efficient protocol for medium throughput fragment screening, hit validation and affinity ranking after single concentration injections was developed. The screening of the fragment library and the good correlation between online estimated pKi values (derived from a single injection) and pKi values measured with a radioligand binding assay (RBA, full range concentration curve) have proven the value of the online fluorescence enhancement assay in FBDD. The online bioaffinity system was also used for rapid hit exploration using single point injections of combinatorial libraries at 96-well format. This led to the discovery of an optimized hit with micromolar affinity towards the α7 nAChR.

CHAPTER 9:Exploring Fragment Screening and Optimization Strategies Using Acetylcholine-Binding Protein

From a niche area of research that was mainly applied by technology focused research groups in the private sector, fragment-based drug discovery (FBDD) has transformed into a rewarding drug-discovery technology that is applied by almost every major pharmaceutical company. Next to biotech and big pharma, the methodology has also attracted considerable interest from academic research groups that have endorsed fragment-based approaches as a sound scientific approach and an attractive low-cost alternative to high-throughput screening, that enables efficient discovery pathways to novel lead and tool compounds. This chapter describes several studies that were performed in our academic research laboratories and in the labs of our collaborators in which acetylcholine-binding protein (AChBP), a homolog to the ligand-binding domain of Cys-loop receptors, has been used as a robust target to investigate the various aspects of fragment-based approaches, including fragment screening technologies and fragment optimization strategies. Timely concepts such as the combination of structural, kinetic and thermodynamic characterization of ligand-induced conformational changes will be described using this particular target. These studies demonstrate how the fragment-based methodology can be used to increase our understanding of the molecular aspects of ligands and fragments binding to protein binding sites.