Place it at the end of a failed joke sequence, after Nelson's Ha-Ha and before the Wrong Answer Game Show sound, so the laugh sets up the joke and the buzzer closes the bit. Pair it with a single laugh first, then let this line end the exchange without adding another reaction after it.
About this sound
This is a Russian voice clip in which Cool Sam delivers the line 'This is no longer funny' in about 1.4 seconds, using the phrase as a direct response to humour that has gone on too long. It gives a deadpan, awkward feeling, like a joke has worn out its welcome and the room has gone quiet. It works as a dry shutdown for humour that has stopped landing.