Targeting Cellular Cytoplasmic Structures

The clinical symptoms of mitochondrial diseases are highly varied and include seizures, vomiting, deafness, dementia, stroke-like episodes, and short stature. Although there are many types of mitochondrial disorders, four of the most common types are as follows Kearns-Sayre syndrome, Leber s hereditary optic atrophy, MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) and MERRE (myoclonic epilepsy with ragged red fibres). 

Since mitochondria are essential to cell health, mitochondrial diseases tend to be severe but, thankfully, relatively uncommon. Accordingly, the medicinal chemistry of mitochondrial disorders is still in its infancy. There are no truly effective drug therapies for mitochondrial disorders, but several agents have been reported to be of some benefit in some individuals. These agents include ubiquinone (coenzyme QIO), carnitine, and riboflavin. These compounds may assist the ailing mitochondria to better complete their metabolic tasks. However, mitochondrial medicinal chemistry is an area of research in need of additional attention. 

Since mitochondria are energy factories, they are essential to cellular life. This fact can be usefully exploited in drug design to enable selective killing of unwanted cell types. For example, the mitochondria of certain parasites are fundamentally different from those of the host human cells. Accordingly, it is possible to selectively kill such parasites by targeting the biochemical uniqueness of their mitochondria. Certain 4-hydroxyquinoline derivatives are effective antiparasitic agents that use this mechanism. 

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Cytoplasmic serine/threonine protein kinases catalyze the transfer of phosphate groups to serine and threonine residues of target proteins. Serine/threonine kinases have been recognized as the products of protooncogenes (e.g., c-mos, c-raj) or as kinases intimately involved with the regulation of serine/threonine kinase activity by cAMP. Some of these kinases specifically phosphorylate cellular structural proteins, such as histone, laminins, etc. Others phosphorylate still more kinases, resulting in either the activation or deactivation of downstream protein kinases. Specific examples in which serine/threonine kinases elicit specific cellular responses are discussed in this chapter. 

Figure 7.1 Drug targets at the level of cellular structure. The mammalian cell presents a variety of druggable targets. The most important ones are located at the level of the cell membrane. Within the cell, cytoplasmic organelles, such as mitochondria, are beginning to be exploited as potential drug targets. The nucleus, at the center of the cell, is an important target for the development of antineoplastic agents for the treatment of cancer.

The cell nucleus is another important source of druggable targets. Surprisingly, the nucleus is not as important to the survival of an individual cell as are many of the cytoplasmic organelles. A cell can live without its nucleus, it just cannot reproduce. (Mature adult human red blood cells, for example, do not have nuclei.) On the other hand, a cell cannot live without its mitochondria. Therefore, the cell nucleus is an important structure to target when designing drugs for diseases in which one wishes to stop cellular reproduction (e.g., cancer, viral or bacterial infections). 

In contrast to mammalian cells, the membrane of bacterial cells is much more complex and, as in the case of Escherichia coli or mycobacteria, it is asymmetric (see Section 1.2.2). The reason for this is that these small cellular life forms depend on diffusion of nutrients and metabolites. All substrates going in and out of the cell must diffuse through their cell walls. This might be one reasons why the surface area to volume ratio is important for bacterial cell shapes. This ratio is determined by the structure of their outer cell wall. To cross such a barrier, mainly by passive diffusion, chemotherapeutics must have other properties in addition to those necessary for suitable pharmacokinetics in the host, as in most cases, the target of the chemotherapeutics is within the cytoplasm. 

TARGETING Despite the vast complexities of eukaryotic cell structure and function, each newly synthesized polypeptide is normally directed to its proper destination. Considering that translation takes place in the cytoplasm (except for certain molecules that are produced within mitochondria and plastids) and that a wide variety of polypeptides must be directed to their proper locations, it is not surprising that the mechanisms by which cellular proteins are targeted are complex. Although this process is not yet completely understood, there appear to be two principal mechanisms by which polypeptides are directed to their correct locations transcript localization and signal peptides. Each is briefly discussed. 

A number of approaches to fluorescently label cytoplasmic and nuclear structures or organelles in living cells are presently available. They vary considerably in terms of ease of use, how specifically they label the target molecules, and in how much they interfere with cellular function. Direct microinjection of fluorescently labelled antibodies into living cells is a universal and efficient way of labelling cellular targets in vivo. In order to avoid interference with cellular function or non-specific cross-reactions the fluorescent antibodies should be prepared as described in Section 2.1 and be first characterized in vitro before they are introduced into cells. 

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