PCS1055

This is a reasonably selective, and potent (6nM) M4 antagonist - selectivity for M4 is difficult to achieve, so this in itself makes this worth considering. It is well characterised pharmacologically and is a neutral competitive antagonist at the M4 receptor. It shows 30-fold selectivity vs M2, and better against the other muscarinic receptors. However, there are two reasons I cannot unambiguously recommend it as an in vitro probe - one is the acetylcholinesterase activity (20-fold selective, 120nM), and the second is the complete lack of broader selectivity information. The paper somewhat unhelpfully states: "Further bio-activity profiling studies conducted during our studies with PCS1055 did not note any significant potency or activity across a panel of other targets (data not shown). I would advise using it as a secondary probe alongside another compound - see below. In vivo the compound shows reasonable exposure; however its brain exposure at the reported 30mpk ip dose is too low to be useful as described in the paper, probably due to efflux (brain:plasma unbound ratio is <<1). In the absence of other probes, my recommendation would be using this compound alongside one of the probes in this publication from Vanderbilt- https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00363 - the lead compound is potent and more selective vs M2, but does have a liability against sigma-1.

This could be a good in cell probe for the target, however, insufficient selectivity data against unrelated targets has been presented (although there is mention of this being conducted). At the dose tested in vivo, adequate brain exposure is not attained (< cellular IC50) but peripheral exposure greater than/equal to the IC50 is achieved for at least 2 hours. Additionally, no pharmacodynamic data has been reported for the probe. A more recent publication reports probe VU6021625, which may be better suited to the study of M4 receptors since it has been more comprehensively tested for selectivity and in vivo pharmacodynamic effects, and exhibits better CNS penetration.