Current Topics in Medicinal Chemistry - Volume 7, Issue 16, 2007

This issue of Current Topics in Medicinal Chemistry focuses on Fragment-based Drug Discovery Approaches and aims to provide readers with both an overview of major methods of the field as well as offering insights from specific case studies. The poet Browning included “less is more” in his fabled poem to Lucrezia and this “minimalist” philosophy can readily be associated with the recent flurry of interest in fragment-based drug discovery approaches. This is exciting science! The figure below plots the number of references cited when “fragment-based” is entered as a literature search term. Interest is growing and reports of the impact of these new methods in aiding in the identification of clinical candidates against various targets have appeared. For a recent compelling review please see P.J. Hajduk and J. Greer, Nature Reviews Drug Discovery, 2007, 6(3), 211-219, A decade of fragment-based drug design: strategic advances and lessons learned. The issue begins with a contribution by Alex et al. describing the conceptual and computational principles that serve as the theoretical basis for fragment-based approaches. With that as background, Hubbard et al. then present a clear description of a combination of experimental approaches to reducing the broad concepts to an implemented reality. Hesterkamp et al. overviews a variety of biophysical approaches and describes their use of Fluorescence Correlation Spectroscopy techniques with specific application to the discovery of inhibitors of prostaglandin D synthase. Zartler and Mo offer a discussion of the practical aspects of NMR-based fragment discovery. AstraZeneca contributes their approaches to fragment-based lead generation with selected examples. Neumann et al. describe their work with chemical microarrays of fragments and surface plasmon resonance detection methods with application to thrombin and PDE. The interested reader should also be aware of the superb monograph edited by Erlanson and Jahnke entitled Methods and Principles in Medicinal Chemistry (2006), 34 (Fragment-Based Approaches in Drug Discovery). It has been our pleasure to work with the contributors to this issue and with Dr. Allen Reitz and the Bentham team. Enjoy!

Fragment-based drug discovery has proved to be a very useful approach particularly in the hit-to-lead process, providing a complementary tool to traditional high-throughput screening. Although often synonymous with fragment screening, fragment-based drug discovery is a far wider area covering high-throughput screening, fragment screening and virtual screening efforts. The unifying feature of fragment-based drug discovery is the low molecular weight of the hit rather than the approach it originates from. Over the last ten years, fragment-based drug discovery has provided in excess of 50 examples of small molecule hits that have been successfully advanced to leads and therefore resulted in useful substrate for drug discovery programs. To our knowledge, there are currently no marketed drugs that can be attributed to these efforts. However, due to the time scales of drug discovery and development it is likely that over the next few years the number of such examples will increase significantly.

Finding novel compounds as starting points for optimization is a major challenge in drug discovery research. Fragment-based methods have emerged in the past ten years as an effective way to sample chemical diversity with a limited number of low molecular weight compounds. The structures of the fragments(s) binding to the protein can then be used to design new compounds with increased affinity, specificity and novelty. This article describes the Vernalis approach to fragment based drug discovery, called SeeDs (Structural exploitation of experimental Drug startpoints). The approach includes the design of a fragment library, identification of fragments that bind competitively to a target by ligand-based NMR techniques and protein crystal structures to characterize binding. Fragments that bind are then evolved to hits, either by growing the fragment or by combining structural features from a number of compounds. The process is illustrated with examples from recent medicinal chemistry programmes to discover compounds against the oncology targets Hsp90 and PDK1. In addition, we summarise our experience with using molecular docking calculations to predict fragment binding and anecdotes on the selectivity and binding modes for fragments seen against a range of targets.

Novel starting points for medicinal chemistry programmes can be effectively identified by screening libraries of fragment molecules in biochemical assays at high concentration. The key to success with this approach is the combination of a high quality fragment library with sensitive biochemical screening methods. There are an increasing number of literature reports where weakly active fragment molecules have been identified by high concentration biochemical assays. We have successfully demonstrated the use of high concentration screening of fragments, using a portfolio of single-molecule Fluorescence Correlation Spectroscopy (FCS+plus) detection techniques to ensure the highest reproducibility and sensitivity, and have determined the binding mode of active fragments to target proteins by X-ray crystallography. Further biophysical detection methods are reviewed for their applicability to studies of fragment binding.

Fragment-based drug discovery (FBDD) needs a biophysical assay to complement, or even replace, biochemical screening. NMR is the best choice for this because NMR delivers many different types of data that impacts medicinal chemistry decisions. There are a multitude of different NMR methods which can be employed to these ends. The choice of which method to use will be different for every need. We discuss the different methods, the data they produce, and how they are best utilized in a FBDD setting.

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).

Fragment-based screening has recently evolved into a promising strategy in drug discovery, and a range of biophysical methods can be employed for fragment library screening. Relevant approaches, such as X-ray, NMR and tethering are briefly introduced focussing on their suitability for fragment-based drug discovery. In particular the application of surface plasmon resonance (SPR) techniques to the primary screening of large libraries comprising small molecules is discussed in detail. SPR is known to be a powerful tool for studying biomolecular interactions in a sensitive and label-free detection format. Advantages of SPR methods over more traditional assay formats are discussed and the application of available channel and array based SPR systems to biosensing are reviewed. Today, SPR protocols have been applied to secondary screening of compound libraries and hit conformation, but primary screening of large fragment libraries for drug discovery is often hampered by the throughput of available systems. Chemical microarrays, in combination with SPR imaging, can simultaneously generate affinity data for protein targets with up to 9,216 immobilized fragments per array. This approach has proven to be suitable for screening fragment libraries of up to 110,000 compounds in a high throughput fashion. The design of fragment libraries and appropriate immobilization chemistries are discussed, as well as suitable follow-up strategies for fragment hit optimization. Finally, described case studies demonstrate the successful identification of selective low molecular weight inhibitors for pharmacologically relevant drug targets through the SPR screening of fragment libraries.

The most prescribed and top selling drugs of 2006: generics, biologics and, of course, Lipitor™. In the spring of 2007, data for the top 200 prescription drugs, in terms of both sales and number of dispensed prescriptions, was released [1-3]. Total sales of prescription drugs reached $275 billion in 2006, an impressive 8.3% increase from 2005; in contrast, sales only increased 5.3% over 2004. The number of dispensed prescriptions also increased in 2006 to 3.7 million (a 4.6% increase from 2005) [1-3]. These data are very encouraging for the pharmaceutical industry, especially considering that 2006 witnessed the loss of patent protection for blockbusters Zocor™, Zoloft™, Plavix™, Provochol™ and Mobic™ which fell victim to generic competition. In fact, the overriding theme of the 2006 was a significant increase in generic prescriptions and sales. While many factors play a role, the 8.3% increase in sales is primarily due to the successful launch of the full Medicare Part D benefit which extended prescription drug coverage benefits to US citizens that historically had little to no coverage. Medicare Part D encompasses every major class of pharmaceuticals and, in 2006, 63% of all prescriptions dispensed to Medicare Part D beneficiaries were generic products [1-3]. Alarmingly for big Pharma, the number of generic products is growing (the FDA approved 371 new generic products in 2006 and their number is expected to further increase in 2007), and drug plans can be expected to elect lower cost generics in preference to name brand drugs. What is the effect of generic competition for a well established, safe and effective drug? Merck's Zocor™ is a good example. In 2005, the statins Lipitor™ and Zocor™ were ranked No.1 ($7.6 billion) and No.2 ($ 4.5 billion) in sales, and No. 1 and No. 11 in prescriptions dispensed, respectively. After generic variants of Zocor™ entered the market mid-year 2006, Pfizer's Lipitor™ remained No. 1 in terms of both sales ($ 8.6 billion) and prescriptions dispensed (74,020) while Zocor™ slid to No.7 in sales ($3.2 billion) and No. 25 in prescriptions dispensed. It will be interesting to see impact of generic Zocor™ on Lipitor™ sales in 2007, as drug plans steer towards the cost savings of generic statins. Also in 2007, the industry will witness first time generic competition for a number of other ‘blockbuster’ drugs such as Prevacid™ (No. 5 in 2006 sales at $3.6 billion) [1-3]. In 2006, the top 10 products in the US market by sales were: 1) Lipitor™ ($ 8.6 billion), 2) Nexium™ ($ 5.1 billion), 3) Advair Diskus™ ($ 3.95 billion), 4) Aranesp™ ($ 3.94 billion), 5) Prevacid™ ($ 3.5 billion), 6) Epogen™ ($ 3.2 billion), 7) Zocor™ ($ 3.1 billion), 8) Enbrel™ ($ 3.08 billion), 9) Seroquel™ ($ 3.02 billion), and 10) Singulair™ ($ 3.0 billion) [1]. These 10 products accounted for 15% of total sales of the top 200 drugs in 2007, and three of the top 10 (Aranesp™, Epogen™, Enbrel™) are biologics. Indeed, 2006 witnessed an increase not only in generic drug sales and prescriptions dispensed, but also biologics. Amgen's Aranesp™ significantly led all top 20 earners with a 42% increase in revenue over 2005 [1-3]. IMS Health forecasts continued sales growth in the pharmaceutical sector in the 6% to 9% range through 2010 and predict that both generic and biologic sales will grow more rapidly and lead the market [1-3]. How will these emerging trends impact big Pharma, the biotech industry, branded drugs and non-biologic small molecule drug sales? Only time will tell, but there are clear challenges to an already struggling pharmaceutical industry. REFERENCES [1] Lamb, E. Top 200 prescription drugs of 2006. Pharmacy Times, May 2007, pp.1-4, and reference therein. [2] IMS Health. Press release. March 8, 2007, www.imshealth.com [3] For more information on generic drug approvals and impact on sales see the US Food and Drug Adminstration: www.fda.gov.