Cellular assays

If the true pfCj of the molecule is known, then a simple inspection of the plot of log Pe (or Papp in the case of cellular assays) versus pH can often reveal the values of both log Po and log Pabl-... 

A lead is variously defined in the pharmaceutical industry as a compound derived from a hit with some degree of in vitro optimization (potency in primary assay, activity in functional and/or cellular assay), optimization of physical properties (solubility, permeability), and optimization of in vitro ADME properties (microsomal stability, CYP inhibition). Moreover, a lead must have established SAR/SPR around these parameters such that continued optimization appears possible. A lead may also have preliminary PK and in vivo animal model data. However, it is the task of the lead optimization chemist to improve PK and in vivo activity to the levels needed for identification of a clinical candidate. 

Chemical Leads Primary Assay IC50 < 1 pM Selectivity Assay 10-fold selectivity Cellular Assay IC50< 1 pM Acceptable in vitro ADME Acceptable PK Profile Potentially patentable... 

Characterized Hits/Series Primary Assay IC50 < 10 pM Selectivity Assay 5-fold selectivity Cellular Assay IC50< 10 pM... 

A series of triazolophthalazines were recently reported to be PDE2 inhibitors with IC50 values as low as 0.1 nM. In this series, compound 2 demonstrated activity in chemotaxis assays. No data were reported for effects on cyclic nucleotide levels, however, efficacy in cellular assays such as the chemotaxis assay suggests the effect is related to PDE2 inhibition in the cell types studied [12]. Pyridopyrimidines such as 3 were claimed to have IC50 values less than 50 nM [13]. 

Several diverse classes of potent CCR5 antagonists, Figure 7 have recently been reported [2], Scaffolds reported to support high potency against CCR5 in antiviral cellular assays included the aminocyclohexyl (compound 13) [45], 4,4-disubstituted piperidine (compound 14, GSK163929) [46,47], tetrahydro-2/T-1,3-oxazine (compound 15) [48], pyrrolidine (compound 16) [49], cyclopropyl (compound 17) [50] and 2,3-dihydro-lH-indene (compound 18) [51]. 

Different oxidation states of sulfur have also been explored, particularly sulf-ones and sulfonamides as transition state analogs of lysine deacetylation, but without much success. The monodentate SAHA-like methyl sulfoxide 7 proved most potent, but still with an enzyme IC50 of only 48 pM and indications of HDAC/TDAC selectivity in cellular assays [28]. 

Page, J. S., Rubakhin, S. S., and Sweedler, J. V. (2000). Direct cellular assays using off-line capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Analyst 125, 555-562. 

Secondary assays depend on the project. Where the primary screen was a cell-based assay, the secondary assay may be a radioligand competition binding assay. In other cases, such as where the primary screen was a biochemical assay, the secondary assay may be a cellular assay, and may be functional or mechanistic. One of the issues that may arise at this stage is that compounds with reasonable activity in the primary assay may not show activity in the secondary assay. There can be a number of reasons for this, including insufficient potency, inability of the compound to get into cells, or a higher intracellular concentration of the natural ligand (e.g., ATP) if the inhibitor is a competitive inhibitor. It is often necessary at this stage to prepare additional compounds in the series to get compounds of sufficient potency and/or permeability so that cellular activity can be demonstrated. 

As with caspase-3, these hits were converted into potent, soluble inhibitors by replacing the disulfide linkage with a simple alkyl linkage (Fig. 9.10). As in the case of caspase-3, rigidifying the linker could improve affinity, as could introducing a hydrophobic moiety at the S2 position. Several of these molecules demonstrated activity in cellular assays and selectivity for caspase-1 over the closely related caspase-5. Crystallography of several of these molecules in complex with caspase-1 revealed that they bind in an extended conformation as expected, but that the S2 pocket that collapses in caspase-3 remains open in caspase-1. 

The activities of the various C-20 alkyl congeners of VBL in cellular assays of growth inhibition and mitotic arrest are presented in Table I. Only one type of substitution, the 20 -methyl derivatives (3 and 4) of... 

---