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Outrunning the Bear.

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High-Throughput Screening
Outrunning the Bear
Jean-Louis Reymond*
biocatalysis · combinatorial chemistry · drug
design · high-throughput screening · self-assembly
Chemistry is the art of transforming
[*] Prof. J.-L. Reymond
Department of Chemistry and Biochemistry
University of Berne
Freiestrasse 3, 3012 Bern (Switzerland)
Fax: (+ 41) 31-631-8057
Lehn and Whitesides, supramolecular up a sizable portion (several percent) of
chemistry aims at forming complex the DCL at equilibrium.[11] The situation
systems by the self-assembly of molec- is similar with target-accelerated synular building blocks through noncova- thesis.[12]
lent interactions without outside interRecently Kazlauskas, Gleason, and
vention.[5] Noncovalent self-assembly co-workers[13] found a solution to this
from mixtures of molecular building mixture selection problem starting from
blocks provides a paradigm for obtain- yet another area of chemistry, biocataling complex supramolecules not acces- ysis. In biocatalysis one uses enzymes for
sible by stepwise synthesis, and the organic synthesis, in particular for their
concept was also suitable for equilibrat- properties as mild, environmentally
ing components linked by covalent friendly, and highly selective catalysts.[14]
bonds.[6] Compound mixtures were The most commonly used biocatalytic
brought back to the field of drug dis- step is the kinetic resolution of racecovery when it was shown that equili- mates, whereby an enzyme converts one
brating mixtures of building blocks for enantiomer (A) of a racemic substrate
small-molecule inhibitors such as pep- (A, B) preferentially into a new product
tides[7] and imines[8] in the presence of a (P) while leaving the other (B) untarget binding protein resulted in an touched (Scheme 1). The mathematical
enrichment of the tightest binding in- treatment of kinetic resolution as dehibitors. This type of equilibrating mix- scribed by Kagan and Fiaud[15] shows a
ture was termed a dynamic combinato- surprising property: even if the enzyme
rial library (DCL).[9]
is not completely selective for one of the
The dynamism of a DCL comes to enantiomers, the reaction leaves the
play during the equilibration phase in unreacted enantiomer of the substrate
the presence of a target ligand. How- with an optical purity higher than the
ever, this dynamic state is short-lived intrinsic selectivity of the enzyme, that
and inexorably leads to a static state of is, R @ S (Figure 1). This higher degree
thermodynamic equilibrium. This is of purity comes at a cost, which is that
quite problematic because at equilibri- the yield of the pure unreacted substrate
um it is almost impossible to distinguish is less than the theoretical maximum of
between similar compounds, and a small 50 %.
advantage of one library member
over another in binding to the
target results in only a proportionately small increase in its concentration. Therefore, the DCL is
able to select the best library
member for binding to its target
only if its properties are far superior to those of the other library
members.[10] Recent mathematical
models show that a single DCL
Scheme 1. Principle of kinetic resolution. Differentiamember must bind the target tion favoring A over B is achieved either by the catathree to four orders of magnitude lyst (kA < kB, no target) or by differential binding to a
better than the average to make target (KA < KB, nonselective catalyst kA = kB).
Angew. Chem. Int. Ed. 2004, 43, 5577 –5579
DOI: 10.1002/anie.200460313
matter. Although turning lead into gold
does not really work, chemists can
design, select, and synthesize new compounds with exceptional properties, in
particular, drugs for treating diseases.
One critical aspect of drug-discovery
technology is the activity-selection step,
which is necessary because it is not
possible to reliably predict the nature
and magnitude of the molecular interaction between a drug target such as an
enzyme and the drug itself. Selecting for
active compounds therefore must rely
on high-throughput screening, whereby
a reference assay for a given activity is
used to test series of compounds.[1]
The development of high-thoughput
screening was a consequence of the
invention of combinatorial chemistry, a
technology which enabled chemists to
synthesize millions rather than tens of
compounds within a very short time.[2]
The essence of combinatorial synthesis
was to prepare large numbers of compounds by using only a few operations,
such as in the split-and-mix protocol.[3]
These methods involved in part the
handling of compound mixtures, but this
aspect was considered too uncertain and
was set aside, and the field of combinatorial chemistry has mostly concentrated
on developing parallel and solid-phase
synthesis methods for single compounds.[4]
The idea of compound mixtures did
not just end there but soon resurfaced in
a very different context, that of supramolecular chemistry. As defined by
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 1. Ratio of remaining substrates R =
A/B as a function of overall conversion and
selectivity S = kB/kA (selective enzyme) or S =
KB/KA (target binding). conv. = conversion of
substrate A, B to product P; S = ln[(1 c)
(1 ee)]/ln[(1 c)(1 + ee)] = ln[(1 c)
(2/(R + 1))]/ln[(1 c)(2R/(R + 1))]; ee = (A B)/
(A + B). Note that R largely exceeds S at high
survival of the better inhibitor and
elimination of the weaker inhibitors.
After seven cycles of periodic recoupling at 16-hour intervals, only a
single inhibitor remained in excess
of 100:1, even through it was only
2.3 times more potent than the next
library member for binding to carbonic anhydrase. The experiment was
in part lucky because the carboxylic
acid component of the dipeptide does
not interact with the target carbonic
anhydrase and can be allowed to
accumulate in the system.
Scheme 2. Kinetic resolution of dipeptide library
The pseudo-DCL has elements
using pronase.[13] The protease pronase is separat- reminiscent of a living system. The
ed from the target carbonic anhydrase by a dialyfirst element, previously described, is
sis membrane. Kxy denotes the dissociation conmolecular death through hydrolysis
stant of the carbonic anhydrase–dipeptide aa bb
by pronase. The second and third
complex. The best binders to carbonic anhydrase
elements are molecular birth by reamong the dipeptides escape hydrolysis by pronase.
synthesis and an energy source in the
form of the activated esters used to
achieve recoupling. Energy expenditure allows the system to stay away
In the pseudo-DCL experiment the
selection by target binding versus de- from thermodynamic equilibrium, an
struction by pronase can be replayed essential feature of living systems. In
over and over again, and a small pref- comparison to previous DCLs, the key
erence in binding to the target eventu- here is that resynthesis cycles can be
ally results in a large enrichment of the timed precisely to allow the system to
tighter binding compound. Just like two periodically move to high conversion
unfortunate hunters trying to outrun an where selection most favors the best
angry bear, none of the compounds in binders (Figure 1). This human interthe pseudo-DCL can outrun pronase. vention for controlling time will be
Yet outrunning the other library mem- extremely difficult to remove to make
bers by only a small amount in terms of a fully autonomous system.
target binding is sufficient to ensure
Kazlauskas, Gleason, and co-workers recognized that the same equation
would apply when using an unselective
enzyme to destroy a mixture of compounds in the presence of an excess of a
target binding protein.[13] This kinetic
resolution would enhance the ratio of
compounds in excess of their relative
binding constants to the target. The
concept was demonstrated for a mixture
of dipeptides competing for binding to
carbonic anhydrase as a target, using the
protease pronase as the nonselective
enzyme to destroy the weaker binders (Scheme 2). However, as for the
kinetic resolution of racemates, this
selection came at the cost of destroying most compounds, including
part of the tightest binders.
Kazlauskas, Gleason, and coworkers have now used this concept
to produce a self-selecting DCL
setup that delivers only the tightest
binding member in the DCL while
leaving only traces of the others.[16]
The amino ends of the dipeptides,
which are released by the action of Scheme 3. Pseudodynamic combinatorial library of
the pronase enzyme used to destroy dipeptides.[16] The amino ends bb1–j of the hydrounbound inhibitors, are periodically lyzed dipeptide are periodically recoupled by timed
recoupled to activated esters, which additions of activated esters aa -X. The esters are
is separated
allows the regeneration of the difrom the target carbonic anhydrase by a dialysis
peptide library from its components
membrane. In the experiment there are four actiand thus results in a pseudo-DCL vated carboxylic acid esters (i = 4) and two amino
(Scheme 3).
acids (j = 2), forming eight possible dipeptides aaxbby.
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
[1] W. P. Walters, M. Namchuk, Nat. Rev.
Drug Discovery 2003, 2, 259 – 266.
[2] Combinatorial Chemistry: Synthesis and
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2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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