As we mentioned last week, July
is bromodomain month at Practical
Fragments. Today we’ll start by looking at two closely related bromodomains, one found
in cyclic-AMP response element binding protein (CBP) and another from adenoviral
E1A binding protein of 300 kDa (EP300). Both proteins have been implicated in a
variety of diseases, particularly cancer, so a chemical probe would be very
valuable.
Alexander Taylor and
collaborators at Constellation Pharmaceuticals, Genentech, and WuXi, describe
such a probe in a recent paper in ACS Med. Chem. Lett.
The researchers screened about 2000 fragments in a thermal shift assay using
0.8 mM of each fragment. Compounds that increased the melting temperature of
the CBP bromodomain by at least 1° C were validated first by time-resolved
fluorescence resonance energy transfer and then by 15N HSQC NMR,
ITC, and X-ray crystallography. Compound 1 was one of the more attractive hits,
in particular because it was considerably less active against BRD4, whose
inhibition causes all sorts of changes to cells.
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The selectivity of CPI-637 against
other bromodomains is also good (> 700-fold less active against BRD4),
though it does hit BRD9 with sub-micromolar activity. Just as with the initial
fragment, the opposite enantiomer of CPI-637 is considerably less active.
Although no pharmacokinetic data are provided, at the very least this should be
a useful probe for cell-based studies.
Switching gears to another aspect
of CBP, the multidomain protein p300/CBP-associated factor (PCAF) has a
bromodomain that may bind to CBP, though the biology is not entirely clear. PCAF
is known to bind an acetylated HIV protein, and has been proposed as a target
for AIDS. Obviously this is another opportunity for a chemical probe! The first
steps are reported in a paper by Stefan Knapp and collaborators at Goethe
University Frankfurt, University of Oxford, Leiden University, ZoBio, and
University of Cambridge, published in J.
Med. Chem (and open-access).
The researchers screened two
separate fragment libraries using either thermal shift assays (at 1 mM
fragment) or TINS. Hits were confirmed using SPR and crystallography, resulting
in seven structures. As expected, all the fragments bound at the site where
N-acetylated lysine normally binds. The PCAF bromodomain appears to be quite
rigid, with little movement in structures with the different bound fragments. A
few elaborated molecules were tested, with the best showing low micromolar
affinity as assessed by ITC; crystal structures with these molecules are also
reported and deposited in the protein data bank. It will be fun to see whether
their potency can be improved.
We’ll have another post on
bromodomains next week, but first stay tuned later this week for an updated list of fragment-derived drugs that have entered the clinic.
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