In vitro functional assays

Compounds with cis double bonds in the side chain were in general found to be more potent and efficacious than their triple-bond congeners, both in in vivo and in in vitro functional assays [98, 106, 107]. QSAR models have been generated for the compounds with unsaturated [108] and l, l -dimethyl [96] side chains to determine more precisely the pharmacophoric requirements of the receptor. It is postulated that for optimum potency, the side chain must be of a suitable length and flexibility to have the ability to loop back so that its terminus is in proximity to the phenolic ring. The widely used, potency enhancing 1 - and 2 -methyl substituents would be expected to increase the tendency of the side chain to adopt a looped back, rather than an extended conformation. 

Figure 3 Rodent pharmacodynamic effects versus CbjU for 6. Dashed lines represent a twofold separation from the in vitro functional assay EC50 (122 nM, dashed arrow). mSLA, mouse spontaneous locomotor activity mPPI, mouse prepulse inhibition DRC, dose-response curve SD, single dose. (See Color Plate 4.3 in the Color Plate Section.)...

In the future, models will exist which will link constants for in vitro binding to cloned human receptors (Kd), data from in vitro functional assays (IC50) and animal and human in vivo EC50 values. A composite prediction matrix will be applied rapidly and accurately to the process of synthesis of new compounds for phase I testing. 

In the case of HTS, enablement means having a suitable assay system. Producing the assay system, while not trivial, has become an almost indnstrialized process, greatly facilitated by the plethora of available, validated assay types and formats and, in the case of in vitro functional assays, by the extremely low protein purity that is often tolerated (see below). In addition, only limited chemistry resources are typically expended to determine whether the chemical starting points that emerge from HTS are indeed tractable. 

In vitro functional assays—receptor binding enzyme inhibition/induction... 

The target compounds were evaluated in an in vitro functional assay and a binding assay. In addition, they were injected into American cockroaches and tobacco homworms. The compounds were also added to lesion nematode (Protvlenchus spp.) preparations. 

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