Chemistry Reference
In-Depth Information
Agood way to compare ligands with different affinities/potencies and molecular weights is
to normalize the affinity/potency with the ligand size.
[
9, 10
]
This is called ligand efficiency
(LE), with the following definition:
[
10
]
LE
=−
G
bind
/N
non-hydrogen atoms
=−
RT
ln
K
d
/N
non-hydrogen atoms
(4.1)
where
G
bind
is the free energy of binding,
N
non-hydrogen atoms
is the number of heavy atoms
in the ligand and
K
d
is the dissociation constant. In practice,
K
d
is often replaced with
IC
50
values. Since an orally available drug should adhere to the Lipinski 'rule-of-five',
[
11
]
including a maximum molecular weight (MW) of 500, the ligand efficiency should be at
least 0.29 kcal mol
−
1
assuming a desired potency of 10 nMand that themeanmolecular mass
for a non-hydrogen atom in drug-like compounds is 13.3.
[
10
]
In terms of ligand efficiency,
this compound is then comparable to a fragment witha1mMpotency and 14 non-hydrogen
atoms (MW
400),
both with a ligand efficiency of 0.29 kcal mol
−
1
. For comparison, a study of 160 ligand-
protein complexes showed that the maximum binding affinity per heavy atom for organic
compounds is approximately 1.5 kcal mol
−
1
.
[
12
]
Extending the ligand efficiency concept,
the polar surface area has also been used as the normalizing factor instead of the number
of non-hydrogen atoms.
[
13
]
Compared with biochemical assays used in HTS, there are considerable advantages to
using biophysical binding techniques such as NMR. The superior control of experimental
parameters and the state of the sample components minimizes artifacts. Also, the sample in
itself is less complex than in a typical HTS assay well. When using an NMR technique as a
binding assay, a good practice is always to collect also a reference
1
H 1D spectrum in which
it will be immediately obvious (i) if the compound (or an important buffer component) for
some reason is not present in the solution, (ii) if the compound is pure and intact or not or
(iii) if the target macromolecule has unfolded, aggregated or precipitated.
[
14
]
An additional
attractive feature is that it is possible to start the fragment screening campaign as soon as
1-2mg of the target has been produced since generic binding assays are used. No time-
and resource-consuming assay development and formatting are necessary. A spin-off from
an early fragment screen could also be identification of reference compounds to be used as
tools in an HTS assay development.
When developing the weakly but efficiently binding fragment hits, it is important to
ascertain that each feature added to the fragment contributes substantially to the binding
energy. Structural information on fragment interactions with the target, preferably high-
resolution crystal or NMR complex structures, will dramatically increase the chances of
developing the fragment hits to potent compounds while keeping the ligand efficiency
high.
[
4
]
Due to the high solubility of the fragments, the probability of obtaining crystal
structures of the fragment-target complex is high. Experience has shown that fragments
bind specifically and give well-resolved electron densities. Hits from HTS campaigns may,
of course, be both highly potent and efficient binders, but very often the hit-to-lead pro-
cess starts off with relatively large molecules with potencies in the high nM to low M
range and low to moderate ligand efficiencies. The development of such hits will in gen-
eral require more synthetic chemistry resources than in the more focused fragment-based
screening approach. It is difficult to identify the parts of the larger molecule responsible for
the important interactions without synthesizing a large number of molecules and testing
them. Fragments are also less likely to contain interfering moieties that block an otherwise
200) or a compound with a 1 M potency and 28 heavy atoms (MW
≈
≈
