Covalent fragment-based lead
discovery is becoming ever more popular, driven by
success against difficult
targets such as KRAS
G12C. These efforts require the design of new
libraries, and in a recent
J. Med. Chem. paper Simon Lucas and
colleagues at AstraZeneca describe their design philosophy. (Co-author Henry Blackwell
presented some of this work at the CHI FBLD meeting earlier this year.)
AstraZeneca has taken great care
in building their fragment libraries; we discussed the revamp of their general
fragment library as well as a “low HBD” (hydrogen bond donor) library
here and
here. For their covalent library, they considered several design features.
First, given that any warhead will add molecular weight (four non-hydrogen
atoms and a hydrogen-bond acceptor for an acrylamide), larger molecules are
necessary, which requires relaxing the
rule of three. Indeed, the researchers
refer to their library as “lead-like.”
Because larger fragments are more
complex, more are needed to explore chemical space. The researchers have built
their library to 12,000 compounds, larger than the typical respondent from our
poll
last year. They have also chosen compounds to be maximally diverse rather than
including near neighbors.
Attractive covalent hits make
specific interactions with a protein target; warheads that are too “hot” can
react non-specifically, as is the case with certain
PAINS. Thus, the researchers chose molecules
having moderate reactivity with the biologically relevant nucleophile
glutathione (GSH).
The design principles are
summarized as:
- Molecular weight 250-400 Da
-
cLogD 0-4
-
GSH t1/2 > 100
minutes
-
Propensity for molecular
interactions (such as hydrogen bond donors and acceptors)
-
Diversity
-
No diastereomeric mixtures
(racemates are OK)
-
Synthetically tractable
-
Purity > 85% (and stable)
These criteria were used to select
~700 historical compounds from within AstraZeneca’s collection. Next, the researchers
chose amines from their internal collection and capped these with an acrylamide
moiety, leading to an additional 1200 molecules. They then turned to custom synthesis
of scaffolds that were under-represented, commercial compounds, and covalent
warheads besides acrylamides, such as cyclic
sulfones. The final library consists
of 88% acrylamides. Molecular weights range from 150 to 420 Da, and compounds
contain 1-6 HBAs, 0-3 HBDs, and 1-3 rings.
The paper briefly describes a
screen against Bfl-1 (or BFL1), a difficult oncology target we
wrote about earlier this year. The protein contains a cysteine residue in the biologically
important BH3 binding site, and previous research by others had identified covalent
binders.
The AstraZeneca researchers tested
Bfl-1 against an early version of the library having just 1400 compounds, which
were incubated at 20 or 200 µM for 24 hours at 4 °C before analyzing by intact protein mass spectrometry. Hits were defined as giving
>50% single labeling and that could be competed with a peptide derived from
the binding partner BIM. Six hits are shown in the paper, with
kinact/KI
values ranging from 0.7 to 9.5 M
-1s
-1, comparable to some
of the early KRAS
G12C hits. Further development of these molecules
is described in a pair of papers that will be the subject of my next post.
Including Bfl-1, the library has
been screened against 15 targets using mass spectrometry, typically
yielding 1-2% hit rates defined as at least 20% labeling of a single site. Given
this record of success, if you’re contemplating building a covalent library,
this paper is well worth studying.
