Use of allosteric targets in the discovery of safer drugs

Abstract

The need for drugs with fewer side effects cannot be overemphasized. Today, most drugs modify the actions of enzymes, receptors, transporters and other molecules by directly binding to their active (orthosteric) sites. However, orthosteric site configuration is similar in several proteins performing related functions and this leads to a lower specificity of a drug for the desired protein. Consequently, such drugs may have adverse side effects. A new basis of drug discovery is emerging based on the binding of the drug molecules to sites away (allosteric) from the orthosteric sites. It is possible to find allosteric sites which are unique and hence more specific as targets for drug discovery. Of many available examples, two are highlighted here. The first is caloxins - a new class of highly specific inhibitors of plasma membrane Ca²⁺ pumps. The second concerns the modulation of receptors for the neurotransmitter acetylcholine, which binds to 12 types of receptors. Exploitation of allosteric sites has led to the discovery of drugs which can selectively modulate the activation of only 1 (M1 muscarinic) out of the 12 different types of acetylcholine receptors. These drugs are being tested for schizophrenia treatment. It is anticipated that the drug discovery exploiting allosteric sites will lead to more effective therapeutic agents with fewer side effects.

Fig. 1

A schematic representation of allosterism. The orthosteric sites are the sites for binding of the substrates or competitive inhibitors of enzymes and agonists or competitive antagonists of receptors. Allosteric sites are away from these sites but their binding to the protein can change its conformation. Binding of positive allosteric modulators leads to either an increase in the affinity of the substrates/agonists or it speeds up the subsequent actions. Negative allosteric modulators act as inhibitors by either altering the affinities at the orthosteric sites or by preventing changes in the protein conformation needed for the subsequent steps.

Fig. 2

Extracellular domains of PMCA as potential allosteric sites. a Reaction cycle of PMCA. PMCA can exist in 2 conformational states: E1 and E2. Transition between these states is mandatory for its ability to pump Ca²⁺ out of the cells. b E1 and E2 differ in their structures. The orthosteric sites for binding to ATP, high-affinity Ca²⁺, release of ADP and inorganic phosphate and for modulation by calmodulin and protein kinases are all on the cytosolic side [figure taken with permission from ref. [31]]. E1-E2 transition results in a change in the conformations of the extracellular domains which are circled. c Extracellular domain 1 sequences of PMCA1 to PMCA4 differ from those of any other ATPases - the sequence of the extracellular domain 1 of Na⁺-K⁺-ATPase is shown for comparison and it has no similarities with the corresponding sequences of PMCA1-PMCA4. Even the sequences of PMCA1-PMCA4 differ from each other.

Fig. 3

Inhibition of PMCA1-PMCA4 by the PMCA4-specific caloxin 1c2 [based on data from ref. [24]].

Fig. 4

Subtype sequence conservation in the muscarinic receptor subtypes. The sequences for the orthosteric site for acetylcholine binding are highly conserved. There are very few sequences which are clearly different between M1 and M5 and these can be used as potential allosteric targets. TM = Transmembrane domain [taken with permission from ref. [45]].

Fig. 5

The allosteric modulator increases the affinity of M1 receptors for acetylcholine (ACh) by 21×. Although only one concentration of BQCA is shown here, the determined EC₅₀ value of BQCA for the M1 receptors was 267 ± 31 nM [the figure is based on data from ref. [48]].