Characterising covalent warhead reactivity

Abstract

Many drugs currently used are covalent inhibitors and irreversibly inhibit their targets. Most of these were discovered through serendipity. Covalent inhibitions can have many advantages from a pharmacokinetic perspective. However, until recently most organisations have shied away from covalent compound design due to fears of non-specific inhibition of off-target proteins leading to toxicity risks. However, there has been a renewed interest in covalent modifiers as potential drugs, as it possible to get highly selective compounds. It is therefore important to know how reactive a warhead is and to be able to select the least reactive warhead possible to avoid toxicity. A robust NMR based assay was developed and used to measure the reactivity of a variety of covalent warheads against serine and cysteine - the two most common targets for covalent drugs. A selection of these warheads also had their reactivity measured against threonine, tyrosine, lysine, histidine and arginine to better understand our ability to target non-traditional residues. The reactivity was also measured at various pHs to assess what effect the environment in the active site would have on these reactions. The reactivity of a covalent modifier was found to be very dependent on the amino acid residue.

Keywords: Covalent drug; Reactivity; Warhead.

Graphical abstract

Fig. 1

(a) With a non-covalent drug (left) the concentration must usually be kept above the minimum efficacious dose (purple line) to have an effect. The concentration and target inhibition are directly related. (b) With a covalent drug once the covalent bond has formed it is not necessary to maintain the free drug (dashed line) in the body at high concentrations as the drug is irreversibly bound to the target (solid line) until the target is degraded. In this case the drug concentration is not directly related to target inhibition. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

The kinetic models used to explain covalent inhibition.

Fig. 3

An example kinetic assay.

Fig. 4

Example NMR spectra for the reaction shown in Fig. 3 looking at one of the vinyl protons. Over time the amount of covalent warhead is reduced and this is observed as the area under these peaks decreasing.

Fig. 5

Results from the example kinetic assay.

Fig. 6

The results of measuring the reactivity of covalent warheads against cysteine (blue dots) and serine (red dots). Compound numbers correspond to their number in the graph and are numbered sequentially based on their reactivity with cysteine (1 = least reactive, 16 = most reactive). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

The measured rate constants for selected covalent warheads reacting with potential target amino acids.

Scheme 1

Synthesis of the covalent warheads of interest.

Scheme 2

Synthesis of Boc-Arg-OMe.