The early stages of new drug discovery in the pharmaceutical industry rely on many steps in the identification and optimization of small drug molecules. These include target identification, assay development, high throughput screening (HTS), hit characterization, and medicinal chemistry optimization. A current problem with this approach is that more funds are spent on drug discovery than those returned from the steadily decreasing number of drugs reaching the market. In order to continue down this avenue of discovery, it is essential that new strategies and technologies be developed and adopted to reverse this trend. One way to do so is to identify and work with more novel and highly validated molecular targets, using genomic, proteomic, and reverse chemical genetic efforts (the elucidation of target function through identification of target-specific small molecule ligands, and subsequent study of their phenotypic effects; see [1] and references therein), coupled with developing cheaper and faster HTS technologies. HTS includes both activity- and affinity-based methodologies, and plays a variety of roles in drug discovery. Most commonly, HTS is used in a methodical search for potential drug leads of molecular targets through cell-based or purified protein-based assays [2, 3]. While activity-based screening can be very robust and efficient, allowing interrogation of many thousands of compounds per day against a single target, affinity-based screening can allow for even greater overall efficiency by screening multiple targets against hundreds of thousands of compounds per day. Furthermore, affinity screening techniques in conjunction with mass spectroscopy (MS) can very efficiently characterize and rank order the primary and deconvoluted hits, greatly facilitating hit-to-lead identification.

In the past decade, MS has become an indispensable tool for the pharmaceutical industry at each stage in drug discovery (see Table 4.1 [4]). Primarily, MS has been employed at the drug development stage. However, due to major advances in affinity-based MS technologies, it is readily becoming a common tool for hit identification in the drug discovery process (see Table 4.2 [4]). A common theme

Table 4.1 Major components of drug discovery phase and their challenges. Included from [4] with permission from Wiley Periodicals.

Phase Numbers of Role or opportunity for MS-based methods compounds

Initial lead discovery

Lead optimization

Candidate selection

Start with @106; prefer 10-100 hits

Start with 1-4 hits; expand to 100-1000 by library technology

From a small set of advanced leads, serial synthesis is used to identify final candidate

Limited by massive installed base of other methods. Current paradigm requires only singlepoint estimate of activity, because low-potency hits are expected (therefore, power of MS-based systems may overmatch the task). Mass-based recognition of compounds may be thwarted by isobaric compounds or impurities.

May be optimum location for use of MS; at this stage, there is interest in accurately determining the respective affinities of compounds derived from the initial leads. Requires screening shift to a secondary assay that could introduce a lag time following early screening phase.

Limited, as complex mixtures or large numbers of compounds are no longer being assayed; more traditional pharmaceutical methods can be applied. Use of MS methods introduced in earlier phases may continue.

for most of these strategies involves massive screening of large chemical libraries or natural products against molecular target proteins to identify potential lead compounds for therapeutics based on compound-target interactions. However, few methods actually allow for both target and ligand(s) to be screened in solution so as to preserve the natural state of both target and small ligand(s). This chapter will provide an overview of how affinity-based MS in combination with ultrafiltration is used in hit identification in the new drug discovery process. We will give examples from our own work and others to emphasize the impact that affinity-based MS has had in new drug discovery. We will cover the importance of developing rapid, highly efficient ultrafiltration affinity-based hit identification strategies, briefly review the principles of specific MS technologies used in these endeavors, and describe the many ways ultrafiltration-based MS is utilized in affinity high throughput screening in today's pharmaceutical industries.

The recent advent of efficient high throughput affinity-based techniques has greatly impacted the new drug discovery process. Such affinity-based technologies have helped begin to answer crucial questions at the earliest possible stage of drug discovery: (i) is the biological target druggable?, (ii) how structurally diverse is the selection of novel small molecules?, and (iii) do selected compounds allow for rapid structure-activity relationship (SAR) development to get lead com-

Table 4.2 MS-based methods proposed for use in lead discovery. Included from [4] with permission from Wiley Periodicals.



Potential leads






Compounds in dynamic



in a column


equilibrium with

et al., 1998)

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