The drug development process often begins with the isolation of the target enzyme that is either directly causing or supporting the symptoms of the ailment. Enzymes are "working macromolecules", in that they facilitate and catalyze a chemical reaction specific to the particular enzyme family, e.g. the alcohol dehydrogenase enzyme family converts short chain alcohols to hydrocarbons and water. After isolation of the target enzyme, the R&D objective becomes the development or discovery of a drug or small molecule that functions to inhibit the enzyme or stop it from working under typical biological conditions.
In the combinatorial chemistry or "shotgun" approach, high throughput enzyme assays are conducted using pharmaceutical libraries containing thousands of small drug like molecules. Each of these small molecules is a potential drug candidate, and the assay is used to determine whether or not the small molecule shows any inhibitory behavior towards the target enzyme. The combinatorial chemistry approach then serves to reduce the library of thousands of small molecules to a reasonable number of drug candidates showing a high probability of successful enzyme inhibition. It is important to recognize though, that the goal here is to collect a base upon which to build, not to isolate the final shelf product. The ultimate deliverable will generally be a derivative or a special formulation, developed through the expenditure of additional research time and resources, of one of the small molecules isolated in the combinatorial chemistry step.
The advantage of the combinatorial chemistry approach over a "therapeutic by design" approach, wherein the drug is designed and developed from scratch using only the ailment and the target enzyme as background sources, is obvious. The shotgun approach allows the scientist to build upon earlier accomplishments, rather that re-inventing the wheel with every new drug project. One of the downsides to the combinatorial approach however, is the discovery that often times the reduced list of potential drug candidates contains molecules that display very strong inhibitor behavior, but lack the other characteristics required for a good drug candidate. These false hits are known as promiscuous inhibitors.
Promiscuous inhibitors are small molecules that function as highly effective enzyme inhibitors, but exhibit non-drug-like traits such as poor specificity and uncorrelated structure-function relationships. Since enzyme specificity and an understanding of the structure-function behavior of the drug is essential to successful clinical trials and subsequent FDA approval, any investiture of time or resources on these promiscuous inhibitors hinders the drug development process and ultimately increases the cost of the final pharmaceutical product.
While the inhibition mechanism for promiscuous inhibitors is still unknown, what has been observed is that the inhibitory behavior is often proceeded by aggregation of the small molecule. In other words, the promiscuous inhibitor is only active as an inhibitor when it is aggregated, and not when it’s in a dissociated (non-aggregated) state. Aggregation is generally concentration dependent. So an understanding of the concentration dependent aggregation behavior of the inhibitor is crucial for early identification and removal of promiscuous inhibitors from the list of promising drug candidates.
Dynamic light scattering (DLS) has proven to be a valuable technique for identifying promiscuous inhibitors. DLS is quick, non-invasive, can work with micro-liter sample volumes, and is highly sensitive to particle aggregation. The figure below shows the DLS size distributions obtained for a promiscuous inhibitor at 8, 16, 20, 40, 60, 80, and 100 mM concentrations. This particular inhibitor was isolated by a customer of Malvern Instruments, and for pending patent reasons, neither the company nor the compound can be revealed here. Sizing measurements were collected with a Malvern High Performance Particle Sizer (HPPS). Samples were prepared in deionized water, and at least 3 measurements were collected for each concentration to insure reproducibility. As evident from the size distributions, the compound is aggregated at concentrations ³ 8 mM. At lower concentrations the size of the particle is too small to be resolved from the background signal, suggesting the absence of aggregation.
Figure 1: Concentration dependence of the aggregation state of an isolated promiscuous inhibitor, characterized with a Malvern Instruments HPPS.
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McGovern, S.L.; Caselli, E.; Grigorieff, N.; Shoichet, B.K. "A common mechanism underlying promiscuous inhibitors from virtual and high throughput screening", J. Med. Chem. 2002, 45(8), 1712.
Pharmaceutical Drug Development: Screening For Promiscuous Inhibitors, Malvern Instruments application note available at www.malverninstruments.com.
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