Biomedical Engineering Reference
In-Depth Information
FIGURE 17.4
Primary HTS and hit triaging.
assay. Results from the primary assay and the subsequent hit triaging through a range
of secondary assays are described in Figure 17.4. The primary HTS was conducted
using the rat
-cell line INS-1E [26] in the presence of a cocktail of cytokines. ATP
levels were determined as a surrogate for cell viability, and compounds increasing the
normalized ATP levels by greater than 75% in this assay were considered hits. A total
of 294 compounds (a hit rate of 1.5%) were identified as hits from the DOS collection,
while 1959 compounds (a hit rate of 0.6%) scored as hits from the MLSMR collec-
tion. The stereochemical structure-activity relationship from the primary screen of
the S N Ar library is shown in Figure 17.5. The 5 R ,6 R configuration was required for
activity at the internal stereocenters. However, the external (2 R or 2 S ) configuration
was less sensitive for activity. Analogs with urea substitutents on the aromatic ring
were favored over sulfonamides and amides. At the secondary amine position, sulfon-
amides were preferred over ureas and amines. This SAR and SSAR coming directly
out of the primary HTS was attributed to the upfront design of the library.
All of the hit compounds were retested in the primary assay at dose, and compounds
with EC 50 <
M were advanced to a total of five secondary assays (counter
screen and functional assays). Fifty-four compounds from the DOS collection and 19
compounds from the MLSMR collection were advanced to these secondary assays.
Unfortunately, all the compounds from the MLSMR collection dropped out during
the secondary assays. However, one DOS compound had a favorable outcome from
this panel of secondary assays and was prioritized for medicinal chemistry efforts
before evaluation of glucose-stimulated insulin secretion in primary human islet cells.
Having established a clean SSAR, more traditional structure-activity relation-
ship (SAR) studies were undertaken to optimize potency. Three different regions of
SRR-14 (Table 17.1) were explored, and ultimately a more potent analog SRR-15
(Figure 17.6) was identified when the p -methoxyphenylsulfonamide was switched to
a benzodioxin based sulfonamide. The optimized SRR-15 was characterized further
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