Combinatorial Chemistry and Drug Discovery

Introduction and Background:

Biology and chemistry interact in many ways. The two fields affect one another greatly, and experts in each field often work in the other. The reason for this interaction and influence is the success each has had in increasing our understanding of the other.

For instance, all of the components of living organisms are molecules, and these in turn are made up of atoms. The dizzyingly complex interactions of these many molecules lead to the functions and behaviors of living systems. While the study of living systems is surely biology, the study of interacting atoms and molecules is undoubtedly chemistry. As a result, chemists have been drawn to the study of these wonderfully complicated chemical systems, and they have made discoveries that have advanced our understanding of biological systems tremendously.

Biology has had a similar impact on chemistry. Chemistry is chiefly concerned with reactions, with transforming one substance into another. Despite great advances in chemical understanding of reactions, there remain reactions that chemists cannot perform under reasonable conditions. There exist, however, biological systems that perform such reactions routinely under mild conditions. By studying these systems, biologists and chemists have learned of reaction pathways and molecular interactions they never could have anticipated.

One of the areas in which there is the most interaction between chemistry and biology is drug discovery. The very nature of a drug requires consideration of both chemistry and biology since it is, by definition, a chemical interacting with a biological system to effect some change. The way drugs are discovered has changed dramatically in the past 10-15 years due to the development of a new methodology for synthesis called combinatorial chemistry. In this experiment, we aim to expose you to this method and express its usefulness in drug discovery.

Combinatorial chemistry is a general method for discovering molecules that possess a desired property. The property can be almost anything: electrical conductivity or semi conductivity, an attractive color, a sweet odor, the ability to block pollen from binding to a histamine receptor, the ability to kill cancerous tumor cells, the ability to kill bacterial cells. Obviously in drug discovery, the latter types of properties are of interest. What distinguishes combinatorial chemistry is its ability to screen many (thousands to millions) of new compounds for a desired property.

Historically, the way compounds with desired properties were discovered and developed was by finding some "lead compound" in nature that exhibited the desired property. The chemical structure of lead compounds were then modified using chemical reactions in order to optimize the property. This approach has proven effective (it is responsible for most of the drugs currently available), but it is very slow since each compound is synthesized individually.

Combinatorial chemistry changes the paradigm for discovery by introducing the notion that compounds should be synthesized as mixtures. When mixtures are produced in chemical reactions, the number of compounds produced increases exponentially. For example, consider a reaction that joins two types of molecules, A and B, to form a molecule A-B. If, as has historically been done, one molecule of each component is combined in a reaction, one product molecule results: A1 + B1 ® A1-B1. If, however, two of each component is included in the same reaction vessel, four product compounds are produced: A1 + A2 + B1 + B2 ® A1-B1 + A1-B2 + A2-B1 + A2-B2. If three of each component is included in the same reaction vessel, nine product compounds are produced, and so on. By combining more and more components in these types of reactions, huge mixtures (or "libraries") can be produced.

The number of tests for the desired property is greatly reduced if those tests are carried out on mixtures of compounds instead of individual compounds. (It is much simpler to perform an antibiotic screen on one mixture of 1000 than on 1000 individual compounds.) Once it has been determined that a mixture has the desired property, the problem changes to identifying which of the many compounds in the mixture is the active one. The process of making that determination is called "deconvolution." There are many methods for deconvoluting an active mixture of compounds, each with advantages and disadvantages. Regardless of the specific method employed, it is possible to identify active compounds performing fewer chemical reactions and fewer tests for the desired property than if the compounds were synthesized and tested individually.

Identification of an antibiotic using combinatorial chemistry:

In this experiment, you will produce libraries of compounds based on the A-B model discussed above. You will simultaneously generate these libraries, test them for antibiotic activity, and deconvolute them to discover which individual compound has antibiotic properties.

The chemical reaction that forms the basis for this reaction is the condensation of an aldehyde (A) and a hydrazine (B) to form a hydrazone (A-B) and water. It is not crucial to understand the chemistry going on in this reaction in order to appreciate the combinatorial method. What is crucial is the understanding that two components come together to form one product.

The parts of the molecules labeled with "R" and numbers represent the variable parts. They can be almost any grouping of atoms, so long as they do not interfere with the reaction shown above. A shorthand for describing the reaction above is to say A1 + B2 ® A1-B2.

In this experiment, three aldehydes (A) and three hydrazines (B) will be combined in a combinatorial procedure to produce 9 hydrazones (A-B). The structures of the six components are shown below.

You will use the cup agar diffusion method to screen the mixtures for antibiotic activity. This method entails the growth of a "lawn" (a uniform layer) of bacterial cells on a solid support (agar). You will bore cups in the agar which can fit about 2 drops of a solution of your mixtures. The solutions will diffuse through the agar about 2 cm from the cup. If the solutions contain a compound with antibiotic activity, there will be no growth in a circle surrounding the cup.