Discovery of Highly Potent AChE by In Situ Click Chemistry

The target-guided click chemistry approach was first tested with acetylcholine esterase (AChE) (Fig. 15.4). The enzyme plays a key role in neurotransmitter hydrolysis in the central and peripheral nervous system [55, 56]. It has two separate binding sites on either end of a narrow gorge [57]. For fragment assembly by the

326 | 15 Click Chemistry for Drug Discovery Step 1. Identification of Anchor Molecule

326 | 15 Click Chemistry for Drug Discovery Step 1. Identification of Anchor Molecule

In situ click chemistry.

In situ click chemistry.

enzyme, the thermal 1,3-dipolar cycloaddition reaction between azides and acetylenes [4] was employed, since the reaction is extremely slow at room temperature and the reactants do not react with the protein. The fragments were designed to bind to either the peripheral anionic site or the active center. They carried azide and acetylene groups, respectively, via flexible spacers. From a total of 52 possible reagent combinations, the enzyme selected only four pairs, leading to the formation of four reaction products in a highly regioselective fashion. These compounds turned out to be more potent than any other non-covalent organic AChE inhibitor, the most potent one having a dissociation constant kd of 99 fM in the case of eel AChE (Fig. 15.4, left).

Additional experiments demonstrated that this fragment-based approach is suitable for the discovery of novel AChE inhibitors from reagents that were not previously known to interact with the enzyme's peripheral site. Thus, a total of 24 acetylene reagents were incubated in sets of up to ten compounds with AChE and the active site ligand, tacrine azide (Fig. 15.4, right) [58]. The triazole products, formed by the enzyme, were identified by HPLC-MS analysis of the crude reaction mixtures. The enzyme selected only the two phenyltetrahydroisoquino-line building blocks that were in the reagent library and combined them with the

In situ click chemistry with acetylcholine esterase (AChE).

In situ click chemistry with acetylcholine esterase (AChE).

tacrine azide within the active center gorge, to form multivalent inhibitors that simultaneously associate with the active and peripheral binding sites. The winning combination involved reagents that have relatively weak binding affinities for AChE (7.8-34 |M), demonstrating that this target-guided strategy is successful even with micromolar binders. The new inhibitors are not only more "drug-like" than the previous phenylphenanthridinium-derived compounds, due to the lack of permanent positive charge and aniline groups and fewer fused aromatic rings, but they are also three times as potent. With dissociation constants as low as 33 fM, these compounds are the most potent non-covalent AChE inhibitors known.

Recent work in the Kolb and Fokin laboratories has shown that the target-guided lead discovery method also works with targets other than acetylcholine esterase, e.g. other enzymes such as carbonic anhydrase II and HIV protease [59], and non-enzymes, such as transthyretin.

Carbonic anhydrases are Zinc-enzymes that catalyze the interconversion of HCO3- and CO2. They are involved in key biological processes related to respiration and the transport of CO2/HCO3, bone resorption, calcification and tumori-genicity [60]. Systemically and topically administered carbonic anhydrase inhibitors have long been used to control the elevated intraocular pressure associated with glaucoma [61, 62]. Most inhibitors are aromatic or heteroaromatic systems that carry a sulfonamide functional group [63-65], which coordinates the Zn2+ ion in the active site. Based on this information, Kolb et al. designed 4-ethynylbenze-nesulfonamide as an anchor molecule for the target-guided formation of carbonic anhydrase inhibitors (Fig. 15.5) [53]. The benzenesulfonamide anchor molecule was incubated with each of 24 azide reagents in the presence of bovine carbonic anhydrase II for 40 h. Analysis of all 24 reaction mixtures by liquid chromatography with mass spectrometry/selected ion mode detection (LC/MS-SIM) revealed

Library of azide reagents (high |jM to mM affinity)

Was this article helpful?

0 0
Peripheral Neuropathy Natural Treatment Options

Peripheral Neuropathy Natural Treatment Options

This guide will help millions of people understand this condition so that they can take control of their lives and make informed decisions. The ebook covers information on a vast number of different types of neuropathy. In addition, it will be a useful resource for their families, caregivers, and health care providers.

Get My Free Ebook


Post a comment