Applying Small Molecules in Biology

Extracts from natural sources have been used in the treatment of diseases since ancient times, but in the early nineteenth century, isolated natural products became available and have ever since been used as biological probes and drugs. The first isolated natural products used as drugs were alkaloids such as morphine, quinine, and codeine (see Chapter 6), and later derivatives of natural products, such as acetylsalicylic acid, were applied. A general feature, however, has been that small molecules have been used either in a random manner or at best in a case-by-case fashion, therefore Schreiber and others decided to systematize the application of small molecules in studies of proteins. This has led to the conception of chemical genetics.

4.2.2.1 Chemical Genetics

Chemical genetics is a research method that uses small molecules to perturb the function of proteins and does this directly and in real time, rather than indirectly by manipulating their genes. It is used to identify proteins involved in different biological processes, to understand how proteins perform their biological functions, and to identify small molecules that may be of therapeutic value.

The term "chemical genetics" indicates that the approach uses chemistry to generate the small molecules and that it is based on principles that are similar to classical genetic screens. In genetics two kinds of genetic approaches, forward and reverse, are applied, depending on the starting point of the investigation. A classical forward genetic analysis starts with an apparent physical characteristic (phenotype) of interest and ends with the identification of the gene or genes that are responsible for it. In classical reverse genetics, scientists start with a gene of interest and try to find what it does by looking at the phenotype when the gene is mutated. In chemical genetics, small molecules are used to perturb protein function: in forward chemical genetics, a ligand that induces a phenotype of interest is selected, and the protein target of this ligand is identified. In reverse chemical genetics, small molecules are screened for effects at the protein of interest, and subsequently a ligand is used to determine the phenotypic consequences of perturbing the function of this protein. Chemical genetics can therefore be regarded as a fruitful and complementary alternative to classical genetics or to the use of RNA-based approaches, such as RNA interference (RNAi) technology.

A now classical example illustrating the power of small molecules in elucidating protein targets is FK506 (or Fujimycin, Figure 4.4), which is a macrolide natural product structurally related to rapamycin (see Chapter 6), and is currently used as an immunosuppressant after organ transplantation. The target of FK506 was not known, but using FK506 as molecular bait to fish its binding protein from biological samples, a protein was identified, called FK506 binding protein (FKBP). It

FK506 (Fujimycin) Secramine

FIGURE 4.4 Examples of compounds used in chemical genetic studies. FK506 was used to identify its protein target, FKBP. Galanthamine was employed as a template for the DOS of structurally diverse analogs, which lead to the identification of secramine, as an inhibitor of vesicular traffic out of the Golgi apparatus.

FK506 (Fujimycin) Secramine

FIGURE 4.4 Examples of compounds used in chemical genetic studies. FK506 was used to identify its protein target, FKBP. Galanthamine was employed as a template for the DOS of structurally diverse analogs, which lead to the identification of secramine, as an inhibitor of vesicular traffic out of the Golgi apparatus.

was found that the binding of FK506 to FKBP inhibits immune function by shutting down a specific molecular signaling pathway. Another natural product, galanthamine (Figure 4.4, see Chapter 16), which is an inhibitor of acetylcholinesterase (AChE) isolated from certain species of daffodil and is used for the treatment of mild to moderate Alzheimer's disease, was used as a template for the so-called biomimetic DOS; that is, a range of diverse chemical reactions was applied to a scaffold similar to that of galanthamine. A 2527 compound library of galanthamine-like structures was prepared and underwent phenotypic screening, identifying secramine (Figure 4.4), as an inhibitor of vesicular traffic out of the Golgi apparatus by an unknown mechanism. After an extensive effort it was discovered that secramine inhibits the activation of the Rho GTPase Cdc42, a protein involved in membrane traffic.

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