Pros And Cons Of Enzyme Screens

Enzyme-based screens offer advantages due to the measurement of a clearly defined enzyme activity. The development of SAR is more tractable as off-target activities are not confounding (e.g., in whole cell screens the inhibitor must traverse membranes, so permeability and active transport confound potency SAR). Often there is considerable knowledge of active sites or the relevant contact surfaces. In vitro systems allow independent manipulation of conditions to tune the ''sensitivity'' of an assay to detect desired classes of inhibitors. These choices define the downstream follow-up of potential inhibitors. Also an in vitro system permits adjustment of the fluxes in a multienzyme pathway to allow detection of cumulative weak inhibition at several loci and to screen an entire pathway (vide infra). Additionally, cell-free systems allow the ability to prepare a single lot of enzyme and store aliquots for the entire screen, rather than relying on living cells grown in tissue culture or obtained in relatively small batches from whole tissue.

However, in vitro enzyme screens are inherently artificial systems that can only approximate the relevant true physiological state. There is the possibility that key regulatory and feedback mechanisms are missing that affect binding to the target. Also they require some level of isolation and purification of the enzyme from some recombinant or natural source, which always raises the possibility that critical component(s) may be purified away or that contaminants may add undesired activities or mask desired ones. Finally, while new pharmacophores may be found from the increased accessibility of the target in vitro, they may prove to be intractable with regard to permeability properties on the target organism. Historically, this has been the case in antibacterial drug discovery.

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