Biomedical Engineering Reference
In-Depth Information
libraries with multiple dimensions of diversities. Library members are individually
synthesized (e.g., by coupling of a reactive moiety to amino-modified DNA fragment)
and HPLC purified, thus enabling highly reliable quality controls (e.g., MS analysis,
spectrophotometric quantification) and featuring almost 100% display for all library
conjugates [63,92]. Moreover, by means of self-assembly, relatively small ESAC
sublibraries (e.g., a comprising 1000 members) easily yield very large DNA-encoded
repertoires (e.g., a 1,000,000-member ESAC library), retaining the initial high quality
and purity (Figure 11.19a) [92].
ESAC technology can be used in at least three different embodiments (Figure
11.19b): (i) each sublibrary can be used independently for affinity capture and selec-
tion experiments (pseudo-single-pharmacophore format); (ii) a sublibrary can be
annealed with an oligonucleotide displaying a known binder to the target and used
for lead optimization selections (affinity maturation setup); and (iii) two (or three)
independent sublibraries can be assembled to form a combinatorial heteroduplex
(or heterotriplex) library in order to perform panning experiment for the de novo
discovery of bi- or tridentate binding molecules [92].
It is worth noting that in analogy to traditional fragment-based methodologies,
the compounds displayed in ESAC architecture are not covalently linked to each
other. Therefore, the relative flexibility between the adjacent binding moieties may
enable the simultaneous engagement of two distinct nonoverlapping binding sites on
the same target protein surface, yielding to a substantial increase in binding affinity
(chelate effect, Figure 11.20) [92,93].
Initially, microarray-based methodologies have been used for decoding in ESAC
library selection experiments [63,64,94,95]. However, to avoid the loss of connectivity
information between fragments during the readout, a novel deconvolution strategy
compatible with high-throughput-sequencing decoding, in which essentially both
coding regions of the DNA heteroduplexes are transferred on the same oligonucleotide
strand, has recently been developed [96].
In analogy with traditional fragment-based drug discovery methodologies, pref-
erential binding pairs have to be connected into a single drug-like high-affinity
compound. Although determination of the optimal linkage (length, flexibility, and
position) between the selected binding fragments selected by combinatorial medic-
inal chemistry can be relatively cumbersome, Melkko and co-workers successfully
employed ESAC technology in various discovery campaigns, including the de novo
identification of binders [94] as well as the improvement of suboptimal ligands
[92,95].
In particular, ESAC technology has been applied to the identification of a
general class of portable albumin binders based on 4-(
p
-iodophenyl)butyric acid
structures [64]. Such a compound has been used to improve the pharmacokinetic
properties of small organic molecules [97] as well as the tumor uptake of tumor-
targeting antibodies [98]. Additionally, the newly discovered compound, in con-
jugation with various contrast agents (e.g., fluorescein or gadolinium complexes
for MRI applications), was shown to considerably enhance their in vivo imaging
performance [64].
