Fragment-based lead discovery on G-protein-coupled receptors

Introduction: G-protein-coupled receptors (GPCRs) form one of the largest groups of potential targets for novel medications. Low druggability of many GPCR targets and inefficient sampling of chemical space in high-throughput screening expertise however often hinder discovery of drug discovery leads for GPCRs. Fragment-based drug discovery is an alternative approach to the conventional strategy and has proven its efficiency on several enzyme targets. Based on developments in biophysical screening techniques, receptor stabilization and in vitro assays, virtual and experimental fragment screening and fragment-based lead discovery recently became applicable for GPCR targets.

Areas covered: This article provides a review of the biophysical as well as biological detection techniques suitable to study GPCRs together with their applications to screen fragment libraries and identify fragment-size ligands of cell surface receptors. The article presents several recent examples including both virtual and experimental protocols for fragment hit discovery and early hit to lead progress.

Expert opinion: With the recent progress in biophysical detection techniques, the advantages of fragment-based drug discovery could be exploited for GPCR targets. Structural information on GPCRs will be more abundantly available for early stages of drug discovery projects, providing information on the binding process and efficiently supporting the progression of fragment hit to lead. In silico approaches in combination with biological assays can be used to address structurally challenging GPCRs and confirm biological relevance of interaction early in the drug discovery project.     

The use of Xenopus oocytes in drug screening

Introduction: The physiological roles of ion channels are receiving increased interest in both basic research and drug discovery, and a demand for pharmacological approaches that can characterize or screen ion channels and their ligands with higher throughput has emerged. Traditionally, screening of compound libraries at ion channel targets has been performed using assays such as binding assays, fluorescence-based assays and flux assays that allow high-throughput, but sacrifice high data quality. The use of these assays with ion channel targets can also be problematic, emphasizing the usefulness of automated Xenopus oocyte electrophysiological assays in drug screening. Areas covered: This review summarizes the use of Xenopus oocytes in drug screening, presents the advantages and disadvantages of the use of Xenopus oocytes as expression system, and addresses the options available for automated two-electrode voltage-clamp recordings from Xenopus oocytes. Expert opinion: Automated and manual Xenopus oocyte two-electrode voltage-clamp recordings are useful and important techniques in drug screening. Although they are not compatible with high-throughput experimentation, these techniques are excellent in combination or as alternatives to fluorescence-based assays for hit validation, screening of focused compound libraries and safety screening on ion channels with their high flexibility for the choice of molecular targets, quality of data and reproducibility.     

An integrated approach to fragment-based lead generation: philosophy, strategy and case studies from AstraZeneca's drug discovery programmes

Fragment-based lead generation (FBLG) has recently emerged as an alternative to traditional high throughput screening (HTS) to identify initial chemistry starting points for drug discovery programs. In comparison to HTS screening libraries, the screening sets for FBLG tend to contain orders of magnitude fewer compounds, and the compounds themselves are less structurally complex and have lower molecular weight. This report summarises the advent of FBLG within the industry and then describes the FBLG experience at AstraZeneca. We discuss (1) optimising the design of screening libraries, (2) hit detection methodologies, (3) evaluation of hit quality and use of ligand efficiency calculations, and (4) approaches to evolve fragment-based, low complexity hits towards drug-like leads. Furthermore, we exemplify our use of FBLG with case studies in the following drug discovery areas: antibacterial enzyme targets, GPCRs (melanocortin 4 receptor modulators), prostaglandin D2 synthase inhibitors, phosphatase inhibitors (protein tyrosine phosphotase 1B), and protease inhibitors (b-secretase).     

Discovery of β2 Adrenergic Receptor Ligands Using Biosensor Fragment Screening of Tagged Wild-Type Receptor

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Fragment-based drug design: combining philosophy with technology

Fragment-based drug design began more than ten years ago and has been steadily gaining in popularity. This review discusses how fragments have been used to choose druggable targets, and what parameters need to be evaluated if a fragment hit is to be considered a suitable ligand for development. Examples of fragment-based screening from the recent literature are reviewed to highlight the various approaches used, along with the possible application of additional techniques to fragment screening against immobilized targets. Finally, mention is made of two different areas, multi-target drug discovery and selective tumor cell targeting, where fragment-based approaches may play an important role in the future.     

Biofocussed chemoprospecting: An efficient approach for drug discovery

Drug discovery strategies include from broad random screening to focussed target‐based approaches. Structure and substrate information greatly enables target‐based design, but this is limited to relatively few targets; cell‐based screening can identify new targets but often suffers from low hit rates and difficult hit optimization. Thus, newer approaches are needed that can improve the efficiency of screening and hit optimization. Here, we describe an efficient approach for hit generation, which may be called “biofocussed chemoprospecting.” With bio‐likeness and ease of synthesis as priority criteria, libraries may be constructed with good optimization potential, physicochemical diversity, drug likeness and low cost. Following this approach, two libraries based on linear and cyclic dipeptide scaffolds were designed, first as virtual libraries comprising of more than 30000 compounds, and after subsequent filtering, as a small library of a total of 51 compounds. These provided good diversity at low cost, and were tested for bioactivities. The discovery of six active compounds demonstrates a hit rate greater than 10%. This is comparable to target‐based approaches, but the “chemoprospecting” method described here has the additional potential to identify new targets and mechanisms.     

Optimizing the hit-to-lead process using SPR analysis

Secondary screening and lead optimization, where a large number of "hit" compounds are refined to a viable set of "lead" drug candidates, are considered to be bottlenecks to the drug discovery process and are targets for streamlining. Surface plasmon resonance (SPR) is a nonlabel technology that can generate kinetic data on biomolecular interactions. This allows researchers to quantitate the binding characteristics of lead compounds with their targets in terms of affinity, specificity, and association/dissociation rates in parallel. The latest generation of SPR biosensors integrate the hit-to-lead process and generate a greater depth of information, providing answers that cannot be addressed by traditional end-point assays. This allows users to make more informed choices on the selection of candidate molecules prior to preclinical development. A number of studies have used SPR biosensors in secondary screening, lead optimization, quantitative structure-activity relationship analysis, and predictive adsorption, distribution, metabolism, excretion, and/or toxicity evaluations.     

Exploiting high-throughput ion channel screening technologies in integrated drug discovery

Ion channels are increasingly being implicated in disease. Although existing drugs that modulate channel function currently represent a key class of pharmaceutical agents, future ion channel drugs could help to treat an even wider variety of diseases. Despite their disease relevance, ion channels remain largely under exploited as drug targets, chiefly resulting from the absence of screening technologies that provide the throughput and quality of data required to support medicinal chemistry. Although some technical challenges still lie ahead, this historic bottleneck in drug discovery is now being bypassed by newer technologies that can be fully integrated into the early stages of drug discovery and will allow the discovery of novel therapeutic agents. Sequencing the human genome has greatly added to the number of potential drug targets but selecting suitable ion channels for drug discovery research should be based on the potential therapeutic relevance of the channel and not just the availability of suitable screens. Currently, ion channel drug discovery is focused on the need to identify compounds that can provide tractable starting points for medicinal chemistry. Advances in laboratory automation have brought significant opportunities to increase screening throughput for ion channel assays but careful assay configuration to model drug-target interactions in a physiological manner remains an essential consideration. Ion channel screening platforms are described in this review to provide some insight into the variety of technologies available for screening, together with some of their inherent advantages and limitations.     

The use of Xenopus oocytes in drug screening

Introduction: The physiological roles of ion channels are receiving increased interest in both basic research and drug discovery, and a demand for pharmacological approaches that can characterize or screen ion channels and their ligands with higher throughput has emerged. Traditionally, screening of compound libraries at ion channel targets has been performed using assays such as binding assays, fluorescence-based assays and flux assays that allow high-throughput, but sacrifice high data quality. The use of these assays with ion channel targets can also be problematic, emphasizing the usefulness of automated Xenopus oocyte electrophysiological assays in drug screening.

Areas covered: This review summarizes the use of Xenopus oocytes in drug screening, presents the advantages and disadvantages of the use of Xenopus oocytes as expression system, and addresses the options available for automated two-electrode voltage-clamp recordings from Xenopus oocytes.

Expert opinion: Automated and manual Xenopus oocyte two-electrode voltage-clamp recordings are useful and important techniques in drug screening. Although they are not compatible with high-throughput experimentation, these techniques are excellent in combination or as alternatives to fluorescence-based assays for hit validation, screening of focused compound libraries and safety screening on ion channels with their high flexibility for the choice of molecular targets, quality of data and reproducibility.     

Structure-based drug discovery and protein targets in the CNS

Structure-based methods are having an increasing role and impact in drug discovery. The crystal structures of an increasing number of therapeutic targets are becoming available. These structures can transform our understanding of how these proteins perform their biological function and often provide insights into the molecular basis of disease. In addition, the structures can help the discovery process. Methods such as virtual screening and experimental fragment screening can provide starting hit compounds for a discovery project. Crystal structures of compounds bound to the protein can direct or guide the medicinal chemistry optimisation to improve drug-like properties - not only providing ideas on how to improve binding affinity or selectivity, but also showing where the compound can be modified in attempting to modulate physico-chemical properties and biological efficacy. The majority of drug discovery projects against globular protein targets now use these methods at some stage.This review provides a summary of the range of structure-based drug discovery methods that are in use and surveys the suitability of the methods for targets currently identified for CNS drugs. Until recently, structure-based discovery was difficult or unknown for these targets. The recent determination of the structures of a number of GPCR proteins, together with the steady increase in structures for other membrane proteins, is opening up the possibility for these structure-based methods to find increased use in drug discovery for CNS diseases and conditions.     

Structure-based generation of viable leads from small combinatorial libraries

Parallel synthesis and structure-aided design have begun to converge in the process of drug discovery. Virtual screening using X-ray crystal structures of therapeutic targets to front-load a high-throughput screen or to establish a tractable collection for lower throughput assays has become a standard practice in many lead generation settings. The application of similar techniques for increasing the likelihood of including active compounds in a focused combinatorial library is a natural progression that is beginning to be recognized. In this review, we will cover recent reports of small, focused libraries designed for specific therapeutic targets, and for which X-ray crystallographic data is available.     

HTS in the new millennium: the role of pharmacology and flexibility

Over the past decade, high throughput screening (HTS) has become the focal point for discovery programs within the pharmaceutical industry. The role of this discipline has been and remains the rapid and efficient identification of lead chemical matter within chemical libraries for therapeutics development. Recent advances in molecular and computational biology, i.e., genomic sequencing and bioinformatics, have resulted in the announcement of publication of the first draft of the human genome. While much work remains before a complete and accurate genomic map will be available, there can be no doubt that the number of potential therapeutic intervention points will increase dramatically, thereby increasing the workload of early discovery groups. One current drug discovery paradigm integrates genomics, protein biosciences and HTS in establishing what the authors refer to as the "gene-to-screen" process. Adoption of the "gene-to-screen" paradigm results in a dramatic increase in the efficiency of the process of converting a novel gene coding for a putative enzymatic or receptor function into a robust and pharmacologically relevant high throughput screen. This article details aspects of the identification of lead chemical matter from HTS. Topics discussed include portfolio composition (molecular targets amenable to small molecule drug discovery), screening file content, assay formats and plating densities, and the impact of instrumentation on the ability of HTS to identify lead chemical matter.     

Library design for NMR-based screening

Recent advances in NMR-based screening methods have made it possible to screen larger libraries of molecules with higher throughput. However, experience shows that intelligent library design is important if NMR screening is to succeed in aiding our discovery of potent and useful lead compounds. This review presents the current state-of-the-art methodologies for designing primary and follow-up libraries for NMR screening. Diversity, drug-likeness and combinatorial libraries are discussed, and the inherent pitfalls of the NMR approach are addressed.     

Fragment-based lead discovery using X-ray crystallography

Fragment screening offers an alternative to traditional screening for discovering new leads in drug discovery programs. This paper describes a fragment screening methodology based on high throughput X-ray crystallography. The method is illustrated against five proteins (p38 MAP kinase, CDK2, thrombin, ribonuclease A, and PTP1B). The fragments identified have weak potency (>100 microM) but are efficient binders relative to their size and may therefore represent suitable starting points for evolution to good quality lead compounds. The examples illustrate that a range of molecular interactions (i.e., lipophilic, charge-charge, neutral hydrogen bonds) can drive fragment binding and also that fragments can induce protein movement. We believe that the method has great potential for the discovery of novel lead compounds against a range of targets, and the companion paper illustrates how lead compounds have been identified for p38 MAP kinase starting from fragments such as those described in this paper.     

Tools for GPCR drug discovery

G-protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases. The design and implementation of high-throughput GPCR assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates are critical in early drug discovery. Early functional GPCR assays depend primarily on the measurement of G-protein-mediated 2nd messenger generation. Taking advantage of the continuously deepening understanding of GPCR signal transduction, many G-protein-independent pathways are utilized to detect the activity of GPCRs, and may provide additional information on functional selectivity of candidate compounds. With the combination of automated imaging systems and label-free detection systems, such assays are now suitable for high-throughput screening (HTS). In this review, we summarize the most widely used GPCR assays and recent advances in HTS technologies for GPCR drug discovery.     

Fragment-based screening with natural products for novel anti-parasitic disease drug discovery

ABSTRACT

Introduction: Fragment-based drug discovery can identify relatively simple compounds with low binding affinity due to fewer binding interactions with protein targets. FBDD reduces the library size and provides simpler starting points for subsequent chemical optimization of initial hits. A much greater proportion of chemical space can be sampled in fragment-based screening compared to larger molecules with typical molecular weights (MWs) of 250–500 g mol^?1 used in high-throughput screening (HTS) libraries.

Areas covered: The authors cover the role of natural products in fragment-based drug discovery against parasitic disease targets. They review the approaches to develop fragment-based libraries either using natural products or natural product-like compounds. The authors present approaches to fragment-based drug discovery against parasitic diseases and compare these libraries with the 3D attributes of natural products.

Expert opinion: To effectively use the three-dimensional properties and the chemical diversity of natural products in fragment-based drug discovery against parasitic diseases, there needs to be a mind-shift. Library design, in the medicinal chemistry area, has acknowledged that escaping flat-land is very important to increase the chances of clinical success. Attempts to increase sp³ richness in fragment libraries are acknowledged. Sufficient low molecular weight natural products are known to create true natural product fragment libraries.     

NMR fragment screening: tackling protein-protein interaction targets

High-throughput screening of libraries containing compounds of 'drug-like' molecular weight has frequently resulted in no or poor drug candidates, especially when screening against demanding drug targets such as protein-protein interactions. Fragment-based lead discovery and optimization has evolved as a promising solution to this problem by combining the universal adaptability of low-molecular-weight fragments with immediate structural information on fragment binding modes. This review focuses on nuclear magnetic resonance (NMR) fragment screening techniques, which provide a unique combination of medium-throughput, direct binding site information and broad applicability. The utility and exemplary data of chemical shift-detected NMR fragment screening applied to the challenging protein-protein interaction target PDZ domains are summarized.     

High-throughput screening technologies for ion channel drug discovery

Ion channels are attractive targets for drug discovery as an increasing number of new ion channel targets have been uncovered in diseases, such as pain, cardiovascular disease, and neurological disorders. Despite their relevance in diseases and the variety of physiological functions they are involved in, ion channels still remain underexploited as drug targets. This, to a large extent, is attributed to the absence of screening technologies that ensure both the quality and the throughput of data. However, an increasing number of assays and technologies have evolved rapidly in the past decades. In this review, we summarized the currently available high-throughput screening technologies in ion channel drug discovery.     

Advances in capillary electrophoresis and the implications for drug discovery

Introduction: Many screening platforms are prone to assay interferences that can be avoided by directly measuring the target or enzymatic product. Capillary electrophoresis (CE) and microchip electrophoresis (MCE) have been applied in a variety of formats to drug discovery. CE provides direct detection of the product allowing for the identification of some forms of assay interference. The high efficiency, rapid separations, and low volume requirements make CE amenable to drug discovery.

Areas covered: This article describes advances in capillary electrophoresis throughput, sample introduction, and target assays as they pertain to drug discovery and screening. Instrumental advances discussed include integrated droplet microfluidics platforms and multiplexed arrays. Applications of CE to assays of diverse drug discovery targets, including enzymes and affinity interactions are also described.

Expert opinion: Current screening with CE does not fully take advantage of the throughputs or low sample volumes possible with CE and is most suitable as a secondary screening method or for screens that are inaccessible with more common platforms. With further development, droplet microfluidics coupled to MCE could take advantage of the low sample requirements by performing assays on the nanoliter scale at high throughput.