Biomedical Engineering Reference
In-Depth Information
(ii) HPLC purification, mass spectrometry characterization, and mixing of equimolar
amounts of each DNA-conjugate compound to generate a first sublibrary pool; (iii)
splitting of the pool into separate reaction vessels and incorporation of a second
set of building blocks (i.e.,
m
200); (iv) enzymatic DNA tagging (i.e., by stag-
gered hybridization of a partially complementary oligonucleotides and subsequent
Klenow-assisted DNA polymerization) to univocally encode the identity of the last
chemical moiety incorporated; and (v) pooling of the encoded reactions to yield the
final DNA-encoded chemical library.
In 2008, Mannocci et al. first demonstrated the methodology in the construction
of a DNA-encoded chemical library containing 4000 compounds [54]. The library
was spiked with a DNA-oligonucleotide conjugated to D-desthiobiotin, a biotin ana-
log with nanomolar affinity to streptavidin, at the expected concentration of a single
library compound [54,63]. High-throughput-sequencing analysis performed on the
library before and after selection on streptavidin-coated Sepharose beads revealed the
enrichment of the desthiobiotin positive control as well as of other classes of struc-
turally related streptavidin-binding compounds (Figure 11.8) [54,63]. Notably, this
work represents the first implementation of high-throughput-sequencing technology
to decode DNA-encoded chemical library selections [54].
Extending the construction scheme depicted in Figure 11.7 to the use of alternative
chemical reactions (e.g., Diels-Alder, Sonogashira, Suzuki, Mitsunobu) and suitable
building blocks, Buller et al. efficiently synthesized various single-pharmacophore
DNA-encoded chemical libraries (each comprising up to 10
6
DNA-conjugate com-
pounds) [55,57]. Using these libraries, the researchers reported successful appli-
cation of the DNA-encoded chemical library technology to the de novo identifi-
cation of ligands possessing low-micromolar to nanomolar affinities against sev-
eral pharmaceutically relevant target proteins, including human serum albumin
[64], Bcl-xL [65], polyclonal human IgG [54], matrix metalloprotease 3 (MMP-
3) [54], carbonic anhydrase IX [55], TNF
=
[57], and interleukin-2 [66] (see also
Section 11.4).
Moreover, the scientists at Philochem and ETH Z urich demonstrated that single-
pharmacophore DNA-conjugate libraries are also particularly suitable for the affin-
ity optimization of previously discovered lead structures [62]. Such focused DNA-
encoded chemical libraries (also termed
affinity maturation libraries
, as popularized
by phage display) can be rapidly synthesized by incorporation of a known lead struc-
ture during the combinatorial assembly of the library. In 2010, ETH Zurich and
Philochem described the synthesis of a benzamidine-based single-pharmacophore
affinity maturation library, which allowed for the isolation of a
10,000-fold
improved benzamidine-based trypsin inhibitor, with single-digit nanomolar potency
and exquisite selectivity over closely related serine proteases (Figure 11.9) [62].
Iterating the split-pool-split process illustrated in Figure 11.7, it is possible to
generate DNA-conjugate libraries comprising more than two sets of building blocks
and over 10
8
DNA-encoded small molecules [54,55,57,58]. In 2009, Praecis/GSK
accomplished the synthesis of a triazine-based DNA-conjugate library containing
802,160,640 compounds performing four rounds of stepwise pool-split-pool reac-
tions and encoding by DNA ligation (Figure 11.10) [58].
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