REVIEWS

COMPUTERBASED DE NOVO
DESIGN OF DRUGLIKE MOLECULES
Gisbert Schneider and Uli Fechner
Abstract | Ever since the first automated de novo design techniques were conceived only
15 years ago, the computer-based design of hit and lead structure candidates has emerged
as a complementary approach to high-throughput screening. Although many challenges
remain, de novo design supports drug discovery projects by generating novel
pharmaceutically active agents with desired properties in a cost- and time-efficient manner.
In this review, we outline the various design concepts and highlight current developments in
computer-based de novo design.

DE NOVO DESIGN
The design of bioactive
compounds by incremental
construction of a ligand model
within a model of the receptor
or enzyme active site, the
structure of which is known
from X-ray or NMR data106.
Johann Wolfgang
Goethe-University,
Institute of Organic
Chemistry and Chemical
Biology, Beilstein Endowed
Chair for Cheminformatics,
Marie-Curie-Str. 11
D-60439 Frankfurt
am Main, Germany
Correspondence to G.S.
e-mail: gisbert.
schneider@modlab.de
doi:10.1038/nrd1799
Molecular DE NOVO DESIGN produces novel molecular
structures with desired pharmacological properties
from scratch. In this approach, a medicinal chemist
and, equally, de novo molecule-design software is
confronted with a virtually infinite search space. The
number of chemically feasible, drug-like molecules
has been estimated to be in the order of 106010100,
from which the most promising candidates have to
be selected (cherry picked)13. Such a large space pro-
hibits exhaustive searching, despite great advances in
high-throughput screening (HTS) technology. Instead
of the systematic construction and evaluation of each
individual compound, navigation in the de novo design
process relies on the principle of local optimization,
which does not necessarily lead to the globally optimal
solution: the design process converges on a local or
practical optimum. In fact, most software implemen-
tations are non-deterministic, and rely on some kind
of stochastic structure optimization.
Just as chemists with different backgrounds are
likely to propose different molecules as promising
solutions, multiple runs with stochastic de novo
design software will produce different compounds as
a result of the nature of the search algorithm. The
trick is to incorporate as much chemical knowledge
as possible about the structure of the search space
into the design algorithm to facilitate directed navi-
gation towards a goal location. As it is impossible to
enumerate all possible virtual molecules in advance
because of the problem of combinatorial explosion,
only those candidate molecules that represent a local
neighbourhood of the search agent are considered at
a time. The map guiding the search to an optimum is
constructed en passant along the search path, and there-
fore dynamically evolves during the search process.
The virtual search agent mimics a medicinal
chemist, and scoring functions perform a function
analogous to virtual assays. In the ideal case, such an
in silico laboratory provides a road map that guides
the agents to high-quality molecular structures via
tractable synthesis routes. Positive design restricts
this virtual optimization process to small regions
of chemical space that have a higher probability of
containing drug-like molecules. Negative design, by
contrast, defines tabu zones that are characterized by
adverse properties and unwanted structures4,5. Still,
there is no guarantee that a molecule will be retrieved
from chemical space that finds immediate appraisal
from a synthetic chemist. In this context, it is essential
that we learn to accept that de novo design will rarely
yield novel lead structures with nanomolar activity
in the first instance. Rather, the designed structures
will probably represent examples of a prospective new
lead series with micromolar activities that require
further optimization.
The artwork Development II by M. C. Escher pro-
vides an illustration of the concept of chemical space
(FIG. 1). There might exist several activity islands in
chemical space, represented by the well-shaped and
formed lizards; that is, drug molecules with desired

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SCAFFOLD HOPPING
The identification of
isofunctional structures
with different backbone
architectures.
PRIMARY TARGET
CONSTRAINTS
All information that is related
to the ligandreceptor
interaction that is, the
binding affinity of a ligand to
the particular biological target.
INTERACTION SITE
A position in space that is not
occupied by the receptor and in
which a ligand atom favourably
interacts with the receptor.
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Figure 1 | How drug-like chemical space might be
structured. M. C. Eschers Development II The M. C.
Escher Company (Baarn, Holland, 2004). All rights reserved.
pharmacological behaviour. Each island represents a
distinct structural class of molecules that are consid-
ered to be isofunctional with regard to their primary
target. It is impossible to directly hop from island
to island. Instead, one has to take a route through
areas that are populated by less desirable compounds,
depicted by the blurred lizard patterns in the Escher
artwork. With increasing distance from an island
the essential activity-defining molecular patterns
become less pronounced. In other words, SCAFFOLD
HOPPING which might be the desired outcome of a
de novo design experiment to obtain new lead series
with potentially improved properties or to circum-
vent intellectual property constraints depends on a
conceptual abstraction from chemical structure. Any
successful design attempt that aims to generate novel
structures that interact with a given target will have
to be grounded on a representation of molecules that
allows an escape from one activity island to another.
Before reaching a new activity island, however, mol-
ecules will be synthesized and tested that have only
marginal activity; for example, binding constants in
the medium- or even high-micromolar range. Such
candidates would usually not be followed up or might
not even be recognized at all in a drug discovery
project. One reason for this is that wandering through
terra incognita in chemical space can only be guided
by maps that were created from previously existing
knowledge, and extrapolation from this knowledge
requires that small steps be made one at a time.
This review gives an overview of computer-based
molecular de novo design methods on a conceptual level.
We focus on the design of small, drug-like molecules.
There are also attempts in the design of peptides68 and
other polymeric structures911 that are not considered
here. By means of several successful deployments
of de novo design in the hit- and lead-finding stages
of the drug discovery process we demonstrate that
2005 Nature Publishing Group
de novo design provides a method for lead identification.
De novo design can therefore be regarded as a com-
plement to other virtual techniques, such as database
searching, and non-virtual techniques such as HTS.
We also accentuate strengths and weaknesses of current
de novo design approaches. The basic properties of
selected de novo design programs discussed in this
article are given in TABLES 1 and 2.
Concepts
Basically, three questions have to be addressed by a de
novo design program: how to assemble the candidate
compounds; how to evaluate their potential quality;
and how to sample the search space effectively. De novo
design is faced with the problem of combinatorial explo-
sion: the number of different element types and the way
they can be linked together is huge. Furthermore, there
are not only a large number of theoretically possible
topologies but also a variety of conformations for a
single topology. This renders a simple enumeration
of all solutions an exhaustive search impossible.
All algorithmic decisions of a de novo design program
are obviously assessed by the quality of their outcome,
which, in turn, crucially depends on a meaningful
reduction of the search space.
Primary target constraints
What kind of input is necessary with regard to a
particular biological target before a de novo design
run can be started? This question is directly con-
nected to the quality assessment of candidate com-
pounds, because the constraints extracted from the
input are used in scoring the generated structures.
All information that is related to the ligandreceptor
interaction forms the PRIMARY TARGET CONSTRAINTS for
candidate compounds. Such constraints can be
gathered both from the three-dimensional receptor
structure and from known ligands of the particular
target. If the former is consulted, the design strategy
is receptor-based; in the latter case, it is ligand-based.
Receptor-based design starts with the determination
of the binding site. As complementarities in molecu-
lar shape and submolecular physical and chemical
properties are important for specific binding, the
binding site is then examined to derive shape con-
straints for a ligand, as well as specific non-covalent
ligandreceptor interactions in the form of hypo-
thetical INTERACTION SITES. Interaction sites are typi-
cally subdivided into hydrogen bonds, electrostatic
and hydrophobic interactions. Receptor groups
capable of hydrogen-bonding are of special interest
owing to the strongly directional nature of the two
interaction partners hydrogen-bond acceptor and
donor and often form key interaction sites. They
allow the assignation of ligand atom positions with a
complementary hydrogen-bond type within a small
region of space and a defined orientation. Key inter-
action sites have a major role in the effort to reduce
the vast number of possible structures because they
define strong and explicit requirements for successful
receptorligand binding.
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Table 1 | Selected de novo design programs with their basic properties in chronological order (continued in Table 2)
Name (year) Building Primary Search strategy Structure sampling
blocks target
constraints
At Fr Rc Li DFS BFS Rnd MC EA Gr Lk Lat MD Sto Scoring function
HSITE/2D X X X Fitting and clipping of planar Steric constraints and hydrogen
Skeletons12,31,95 skeletons bonds
(1989)
3D Skeletons32 X X X X Steric constraints and hydrogen
(1990) bonds
Diamond Lattice33 X X X X Steric constraints and hydrogen
(1990) bonds
BUILDER v128 X X X X X Steric constraints and key
(1992) interaction sites
LEGEND20 (1991) X X X X Force field
LUDI13,14,9698 X X X X X Empirical scoring function
(1992) (SCORE1; revised version
SCORE2 in 1998)
NEWLEAD30 X X X X X Steric constraints
(1993)
SPLICE60 (1993) X X X X Pharmacophore and steric
constraints
GenStar34 (1993) X X X X Steric constraints and ligand
enzyme contact
GroupBuild18 X X X X Force field
(1993)
CONCEPTS39 X X X X Empirical scoring function
(1993)
SPROUT17,57-59 X X X X X X Solvent accesible surface,
(1993) hydrogen bonds, electrostatic
and hydrophobic interactions
MCSS & X X X X Simplified van der Waals potential
HOOK25,27 (1994) of non-polar interactions
GrowMol21 (1994) X X X X X Simple empirical scoring function
61 X X X X Potential energy
MCDNLG (1995)
At, atoms; BFS, breadth-first search; DFS, depth-first-search; EA, evolutionary algorithms; Fr, fragments; Gr, grow; Lat, lattice; Li, ligand; Lk, link; MC, Monte Carlo
sampling with Metropolis criterion; MD, molecular dynamics; QSAR, quantitative structureactivity relationship; Rc, receptor; Rnd, random; Sto, stochastic.

Derivation of interaction sites by including interaction sites of covalent bonds and

Receptor-based de novo design uses a variety of
methods to deduce interaction sites from the three-
dimensional structure of the binding pocket. HSITE12,
a rule-based method, was the first software specifi-
cally developed for the derivation of primary target
constraints in an automated, de novo design approach.
Only hydrogen-bond acceptors and donors are con-
sidered, but it attempted to identify intramolecular
hydrogen bonds of the receptor. Acceptordonor
pairs that participate in such intramolecular binding
have to be excluded from the list of potential interac-
tion sites. The outcome of an HSITE run is a map of
hydrogen-bonding regions. These regions are centred
at ideal hydrogen-bond geometry and allow a certain
tolerance range of bond length and bond angle around
the centres. The values for the ideal geometry and the
tolerance ranges are empirically derived from crystal-
structure data of small molecules. Other rule-based
methods were developed later that added lipophilic
interaction sites1316. HIPPO17 augmented this concept
bonds to metal ions. Moreover, the idea of complex
hydrogen bonds was introduced: in the case of mul-
ticentred or bifurcated hydrogen bonds, individual
hydrogen-bonding regions were intersected to obtain
complex hydrogen-bonding regions. These regions
are assumed to form particularly strong hydrogen
bonds and are therefore preferred over normal
hydrogen-bonding regions.
Several de novo design programs implement grid-
based approaches for the derivation of primary target
constraints. A grid of points is generated in the binding
site, and interaction energies are computed by placing
different probe atoms or fragments at each grid posi-
tion. An appropriate selection of probes for example,
with hydrogen-bonding capabilities or lipophilic prop-
erties leads to the determination of interaction sites.
Some de novo design programs13,14,18 perform the cal-
culations with the software GRID19, whereas others2023
contain their own implementation of this algorithm.
LigBuilder24 carries out a statistical analysis after the

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grid-based calculations to extract the most promising
interaction sites. The performance of grid-based
methods crucially depends on the resolution of the grid.
It is evident that higher resolution leads to more grid
points and therefore to greater computational costs, so a
compromise has to be made. Two de novo approaches25,26
use Multiple Copy Simultaneous Search (MCSS)27 for
the generation of primary target constraints.
MCSS determines energetically favourable posi-
tions and orientations of functional groups in the

Table 2 | Selected de novo design programs with their basic properties in chronological order
Name (year) Building Primary Search strategy Structure sampling
blocks target
constraints
At Fr Rc Li DFS BFS Rnd MC EA Gr Lk Lat MD Sto Scoring function
Chemical X X X X X Combined score of shape, grid-
Genesis22 (1995) based and scalar constraints
DLD26,99 (1995) X X X X Potential-energy function without
electrostatic interactions
PRO_ X X X X X X Empirical scoring function
LIGAND15,44,100-103
(1995)
SMoG41,42,104 X X X Knowledge-based scoring function
(1996)
BUILDER v229 X X X X Steric constraints
(1995)
CONCERTS35 X X X X Force field
(1996)
RASSE23 (1996) X X X X Force field augmented by chemical
rules
PRO_SELECT16,40 X X X X Empirical scoring function
(1997)
SkelGen63,64 X X X X X Geometric, connectivity and
(1997) chemical constraints
Nachbar45,105 X X X X Target-specific QSAR model
(1998) based on topological connectivity
descriptor
Globus49 (1999) X X X X Molecular similarity based on
all-atom-pairs-shortest-path
descriptor
DycoBlock36,37 X X X X Force field and solvent-accessible
(1999) surface
LEA47 (2000) X X X X Target-specific QSAR model based
on three-dimensional descriptors
LigBuilder24 (2000) X X X X X Empirical scoring function
48 X X X X Molecular similarity based on
TOPAS (2000)
topological pharmacophore and
substructure fingerprints
F-DycoBlock38 X X X X Force field and solvent-accessible
(2001) surface
ADAPT67 (2001) X X X X Weighted sum of DOCK score,
clogP, MM, number of rotatable
bonds and hydrogen bonds
Pellegrini & Field46 X X X X X Target-specific QSAR model
(2003)
SYNOPSIS55 X X X X Two examples: electric dipole
(2003) moment and empirically derived
HIV-RT scoring
CoG50 (2004) X X X X X Molecular similarity based on
fingerprint descriptor
BREED62 (2004) X X X *
*Exhaustive recombination. Exhaustive enumeration; no internal scoring function. At, atoms; BFS, breadth-first search; DFS, depth-first-search; EA, evolutionary
algorithms; Fr, fragments; Gr, grow; HIV-RT, human immunodeficiency virus reverse transcriptase; Lat, lattice; Li, ligand; Lk, link; MC, Monte Carlo sampling with
Metropolis criterion; MD, molecular dynamics; MM, molecular mass; QSAR, quantitative structureactivity relationship; Rc, receptor; Rnd, random; Sto, stochastic.

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Box 1 | Multidimensional optimization
Many practical optimization problems face several objectives at once and drug design
is no exception to this. Typically, optimization techniques reformulate multi-objective
problems as a single objective using a weighted scoring function. Weighted scoring
functions sum the individual objectives and associate a weighting factor to each
objective: f(p) = w1p1 + w2p2 + + wnpn, where wn is the nth weighting factor and pn the
nth property. The setting of the weights is non-trivial. Conflicting objectives give rise
to competition among them. For example, the design of a focused combinatorial
library might be subject to chemical diversity and drug-like physico-chemical
properties88. Such a situation constitutes a pitfall for an optimization algorithm and
can lead to unreasonable solutions. Moreover, the weights specify a single
compromise of the objectives. But there usually exists a whole family of solutions,
each of which corresponds to a compromise in terms of the individual objectives.
The Multiobjective Genetic Algorithm (MOGA)89 parlays the concept of a genetic
algorithm to explicitly incorporate the optimization of several constraints. It attempts
to map out a hypersurface in the search space such that all solutions are considered as
equivalent. The solutions that are part of this hypersurface also termed Pareto
frontier are non-dominated or Pareto solutions. A solution is non-dominated if an
improvement in one objective leads to a degradation in one or more of the other
objectives. In other words, a solution dominates another one if it is equivalent or
better in all objectives and better in at least one objective compared with all other
solutions of the same population. The ranking of a population is inversely
proportional to the number of times a solution is dominated. Solutions can be
inspected to identify the ones that represent the most appropriate compromise of the
individual objectives for the task at hand.
The figure shows potential non-dominated (red circles) and dominated (grey
circles) solutions in a problem with two properties. As indicated by the large arrow
the objective is a minimization
of both properties. Solutions are
non-dominated if the rectangle
formed by lines drawn parallel
1
to the axes contains no other
solution (dotted lines). The non- 2 field to evaluate candidate compounds, but over the
dominated solutions map out
Property 2
0
the Pareto frontier (orange line). 5 The first empirical scoring function in the field of
Numbers next to the solutions 1 2
indicate the number of times 0
they are dominated. The MOGA 1 LUDI13,14. Empirical scoring functions are a weighted
algorithm has been successfully 0 sum of individual ligandreceptor interaction types
applied to a number of problems
in cheminformatics9092 and
recently made its debut in the
field of de novo design50. Property 1
binding site. Multiple copies of functional groups are
randomly placed inside the binding pocket. All frag-
ments are then minimized simultaneously using a force
field such that the forces among individual functional
groups are not considered. Groups are discarded if the
interaction energy between them and the protein is
above a certain threshold. An MCSS run yields a set of
pre-docked fragments that can be further investigated
to choose the most promising ones. The same outcome
can be achieved by the use of docking software, which
is indeed the initial step in some de novo design pro-
grams2830. Rule- and grid-based methods only derive
the primary target constraints: the outcome of these
two methods is a map of the receptor that pinpoints
favourable interaction sites of a ligand. MCSS and the
pre-docking of fragments effectively amalgamate the
derivation procedure and the first steps of structure
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assembly that is, favourable positions of specific
functional groups in the binding site are not only indi-
cated but are already placed at these positions. This
placement of chemical groups provides a starting point
for the next step in de novo design: the assembly of a
complete ligand.
Receptor-based scoring
The evaluation of candidate compounds with scoring
functions is a central task in the design process. The
application of a de novo design program yields more
than one candidate compound. These structures can
either emerge from a single run of the program or from
several runs with one candidate compound per run.
Scoring functions rank the generated structures and
thereby suggest which structures are the most promis-
ing ones. Moreover, during a run they also guide the
design process through the search space by assigning
fitness values to the sampled space.
Conceived approximately 15 years ago, the very
first de novo design programs only applied steric
constraints to guide the search procedure2834, but
more sophisticated approaches emerged in the
following years. Basically, these quality-assessment
approaches can be subdivided into three different
types of receptor-based scoring functions: explicit
force-field methods; empirical scoring functions;
and knowledge-based scoring functions. All of these
approaches attempt to approximate the binding
free energy. Force fields are computationally more
costly than the other two types of scoring functions.
LEGEND20 was the first program that used a force
years quite a few others followed this concept18,23,3538.
de novo design was implemented in the program
commonly supplemented by penalty terms, such as
the number of rotatable ligand bonds. The weights
correspond to the average free-energy contribution
of a single interaction of that type and are obtained by
a regression analysis of a set of receptorligand com-
plexes. Interaction types include, for example, hydro-
gen bonds, electrostatic interactions and hydrophobic
interactions. The regression analysis requires both
known structures and binding constants, and so the
available datasets are limited in size and often feature
similar ligands and receptors. This can result in a bias
of empirical scoring functions towards specific struc-
tural motifs. However, they are fast and have proved
their suitability, and are therefore implemented in
several de novo design programs15,16,21,24,39,40.
Knowledge-based scoring functions have become
popular in the field of docking during the past few
years, yet to date there is only a single de novo design
program, SmoG41,42, that uses its own implementation of
this type of scoring function. Knowledge-based scoring
is grounded on a statistical analysis of ligandreceptor
complex structures. The frequencies of each possible
pair of atoms in contact to each other are determined.
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A Link/grow strategy a Place fragments

N
H O
Ile56
O Link
N
H O

OH
O
O OH
Ki = 16 M
O
O
Phe46 Asp37
b Place first fragment
Define binding pocket Determine interaction sites N
H O

O Grow

OH
O O
O O
B Lattice strategy
N N
H H

OH

O O
O O

Fill pocket with lattice points Find and connect interaction points Assign molecular framework Build molecule
Figure 2 | Principles of structure-based ligand assembly. A | The link (Aa) and grow (Ab) concepts are displayed for the
example of FK506-binding protein (FKBP12) ligand de novo design. On the basis of an X-ray model of the binding pocket
(PDB-identifier: 1fkf), interaction sites were identified. Selected interaction centres are indicated by blue dots (lipophilic), green
(acceptor) and red (donor) lines. A micromolar inhibitor of FKBP12 was designed using the software package LUDI, representing
one of the first successful prospective applications of de novo design93. The magenta-coloured substructures highlight the
linker in the link approach, and the grown part of the molecule in the grow scenario, respectively. B | The lattice strategy
provides an alternative approach that is grounded on a grid representation of potential positions of ligand atoms within the
binding pocket. Lattice points are indicated by grey dots. Ligand candidates are formed from those points that lie along the
shortest path through the lattice connecting interaction sites (the particular molecule shown is an artificial example).

PHARMACOPHORE
The ensemble of steric and
electronic features that is
necessary to ensure the optimal
supramolecular interactions
with a specific biological target
structure and to trigger (or to
block) its biological response106.
QUANTITATIVE STRUCTURE
ACTIVITY RELATIONSHIPS
(QSAR). Mathematical
relationships linking chemical
structure and pharmacological
activity in a quantitative
manner for a series of
compounds. Methods that can
be used in QSAR include
various regression and pattern-
recognition techniques106.
Interactions found to occur more frequently than
would be randomly expected are considered attractive;
interactions that occur less frequently are considered
repulsive. Only structural information is necessary
to derive these frequencies so that a greater number
of structures can be included in the analysis. As the
available structures are also more diverse than those
with known binding affinities, less bias is expected
compared with empirical scoring functions.
Ligand-based scoring
If a three-dimensional structure of a particular biological
target is unavailable but one or more binding molecules
are known, ligand-based design provides an alternative
strategy. This scenario holds true for G-protein-coupled
receptors (GPCRs), which are the most successful drug
targets in terms of therapeutic benefit and potential
sales43. Receptor-based structure generation is inevi-
tably confronted with the problem of conformational
complexity. A ligand-based strategy, in contrast, can
either consider the three-dimensional or the topological
structure of one or more known ligands.
One way to use the information inherent to the
known actives is the derivation of a three-dimensional
ligand PHARMACOPHORE model. Once established, it can
be used to obtain a pseudo-receptor model44. This
facilitates the application of de novo design programs
that were originally developed with a receptor-based
strategy in mind to ligand-based design. Alternatively,
the three-dimensional ligand pharmacophore model

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Binding
Initial state
pocket
Level 1 NH HN
O
HO
Level 2 NH NH NH
N
N O
of-five51. As the proposed molecules are considered
End state NH Designed molecule
O
Figure 3 | Tree model of search space exploration by an automated structure-
generation method. A binding pocket and predicted key interaction sites are given as an
input and are represented by the initial state (root node of the tree). In this example, key
interaction sites are pharmacophore points at which yellow circles indicate sites for a ligand
hydrogen-bond donor and blue circles represent regions for lipophilic ligand parts. Candidate
compounds are assembled by cycles of fragment placing and subsequent scoring of the
resultant partial structures (grow strategy). Partial structures are partial solutions (shown in light
green) if they satisfy the primary target constraints. In the example, some partial structures are
rejected due to boundary violation (Level 1 and 2) or due to mismatching interaction types
(Level 2). Two partial solutions emerge on Level 1. A breadth-first search would follow both
nodes in the search graph simultaneously, whereas the tree shown here selects a single node
for expansion (depth-first search). Selection of this single node could be guided by the score of
the partial solution, by chance or by a combination of both. A path through the search space
tree from the initial state to an end state leads to a candidate compound.
can be used directly in a similarity design method44.
Whereas a (pseudo)receptor guides the design of
structures that are complementary to the primary
target constraints, a ligand pharmacophore model can
be applied to designing structures that are similar to
these constraints. The generalization quality of a three-
dimensional pharmacophore model improves with a
high degree of structural diversity in the set of known
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ligands. A common binding mode of all deployed
ligands is a prerequisite for the building of a substantial
model. A set of known ligands can also be taken as an
input for the development of a target-specific QUANTITA
TIVE STRUCTUREACTIVITY RELATIONSHIP (QSAR) model. The
established model then serves as a scoring function4547.
Another scoring approach is the computation of molec-
ular similarity between a known active compound and
the candidate compounds4850. This requires the choice
of a molecular descriptor and a similarity index. Both
a QSAR model and molecular similarity can be based
not only on the three-dimensional ligand structures
but also on their topological structures. This choice is
made by the selection of appropriate descriptors.
Secondary target constraints
An effectual drug molecule is subject to more objectives
than the binding affinity. Essential drug properties,
such as suitable absorption, distribution, metabo-
lism, excretion and toxicity (the so-called ADMET
properties), clearly emphasize the multi-dimensional
optimization of drug development. Constraints other
than the binding affinity are secondary constraints.
The overall score of structures proposed by a de novo
design program is sometimes calculated as a weighted
sum of the estimated binding affinity and other terms
that directly or indirectly contribute to the sec-
ondary constraints. Candidate compounds with an
increased chance of oral bioavailability can be obtained
by filtering the designed structures with Lipinskis rule-
to be potential leads, a lowered upper bound for the
molecular mass of 350 Da and clogP of 3 might be
more appropriate52. In silico prediction systems are also
able to assist with the avoidance of severe side effects,
such as hERG-mediated sudden death, though their
accuracy is still coarse-grained at best53.
The sole regard of valence rules does not ensure
the generation of chemically reasonable and stable
structures. The issue of unstable chemical groups was
addressed early in the development of de novo design
with a set of chemical rules for example, a list of
undesirable substructures. More difficult to handle,
but also of major importance, is the ease of synthesis,
because the ability to predict binding affinity is still
limited. It cannot be expected from the outcome of a
design study that all the candidate compounds have
the proposed properties. What can be expected is a
significantly increased hit rate compared with the
screening of arbitrary compound collections.
This only underlines the relevance of validation with
biological assays and the consequent need to design
synthetically accessible structures. Synthetic accessibil-
ity subsumes the availability of starting materials and
the synthetic feasibility of the final product by amena-
ble reactions. Even though this issue was recognized
early in the history of de novo design, it was addressed
only recently. The common basic motif of the few de
novo design programs that consider ease of synthesis
is to assemble the building blocks in accordance with
a set of virtual organic reaction schemes.
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For example, the building blocks of TOPAS48 are
obtained by virtual retro-synthesis of a drug-molecule
collection with a set of 11 common organic reactions
the so-called RECAP54 reactions. The same set of
reaction schemes is then used to assemble candidate
compounds. PRO_SELECT 16 adds the template-
substituent idea of combinatorial chemistry. The most
sophisticated approach is undertaken by SYNOPSIS55.
In this program, the building blocks are constituted by
a database of available molecules. Structure assembly
is guided by 70 different simulated organic synthesis
steps so that a synthesis route is proposed for every
generated structure. Furthermore, the acceptance of a
reaction incorporates information about neighbours
of the functional group, other functional groups that
might prevent the reaction, and reactivity rankings
if a functional group is present more than once in a
molecule. An alternative approach is the application
of external software that attempts to assess the syn-
thetic accessibility of a set of candidate compounds
for example, CAESA17 or SEEDS56. These programs
automatically analyse generalized synthetic routes and
select potential precursors from databases of available
compounds. CAESA additionally provides an estima-
tion of the ease of synthesis for example, recogni-
tion of complex ring systems or stereocentres by an
expert rule system.
Structure sampling
The basic building blocks for the assembly of candi-
date structures can be either single atoms or fragments.
Atom-based approaches are superior to fragment-based
methods in terms of the structural variety that can
be generated. But this increase in potential solutions
makes it harder to find suitable candidate compounds
among the ones that are amenable. Fragment-based
design strategies, on the other hand, significantly
reduce the size of the search space. This reduction can
be called a meaningful reduction if fragments are used
that commonly occur in drug molecules. Additionally,
the definition of fragment is variable: a fragment can
be anything from an atom to a polycyclic ring system,
which means that atoms are essentially a subset of
fragments. A composition of building blocks that
contains small fragments and larger ones allows for
structural changes to a different extent depending
on the specific requirements. Many of the early de
novo design strategies were atom-based. But as the
combinatorial problem that is coupled with atoms
as building blocks became more and more evident,
fragments were generally used. Today, we commonly
find building-block sets that are mainly composed of
fragments with more than one atom and padded with
a few single-atom fragments.
There are several general concepts of structure sam-
pling: linking, growing, lattice-based sampling, random
structure mutation, transitions driven by molecular
dynamics simulations, and graph-based sampling.
The linking approach1315,24,25,30,5760 starts with the
placement of building blocks at key interaction sites
of the receptor (FIG. 2). These building blocks are either
656 | AUGUST 2005 | VOLUME 4
2005 Nature Publishing Group
positioned by the de novo program itself or provided
by another program (pre-docked building blocks). The
positioned building blocks are automatically connected
to each other by so-called linkers to yield a complete
molecule that satisfies all key interaction sites. Linkers
are selected with the objective of forming favourable
interactions with the receptor.
The growing procedure1315,18,20,21,23,32,34,41,42,5759 starts
with a single building block at one of the key interac-
tion sites of the receptor (FIG. 2). This starting point is
selected by the program, the user or is given by a pre-
docked fragment. The structure is then grown from the
initial building block in an attempt to provide suitable
interactions for both the key interaction sites of the
receptor and regions of the receptor between two key
interaction sites. PRO_SELECT16 introduced the idea
of combinatorial chemistry to the linking method.
Substituents are linked to a pre-docked scaffold with
user-defined attachment sites. Both the growing and the
linking strategy have their strengths and weaknesses.
The growing strategy can run into difficulties if the
active site contains two or more distinct (sub)pockets
separated by a large gap in which the possible interac-
tions between small-molecule ligands and the protein
are limited18. When fragments are used in combination
with the linking approach, slightly misplaced fragments
or fragments with no strictly defined spatial orienta-
tion can lead to ambiguity during linking. An example
is a phenyl ring, which has no preferred orientation in
a lipophilic binding pocket.
Another concept is the placement of an atomic
lattice in the binding site (FIG. 2). This lattice is made
up of regularly arranged sp3 carbon atoms (diamond
lattice)33, randomly and evenly distributed atoms29, or
pre-docked fragments28. Lattice atoms in the vicinity
of different interaction sites are then joined by find-
ing the shortest path through the lattice atoms. Atoms
that are part of such a shortest path are connected by
newly formed bonds. DLD26 and MCDNLG61 start with
a binding site that is filled with a non-physical arrange-
ment of atoms. The initial atom arrangement is termed
non-physical because atoms are placed or connected
in a way that is non-existent in reality. Examples are
participation of atoms in a number of bonds that far
exceed typical chemical bond valences or which com-
pletely disregard van der Waals radii. Then, an atom
is randomly selected and a randomly chosen transi-
tion is applied to this atom. Such a transition can be a
translation or rotation operation26,61, a change of atom
type or bond type, or the appearance or disappearance
of an atom26. The guidance of iterated transitions by a
potential-energy function finally yields a chemically
valid molecule.
A few de novo design methods implement structure
sampling that is driven by a molecular dynamics simu-
lation. Covalent connections are formed among the
building blocks in a stochastic and reversible manner
to dynamically evolve candidate compounds. Initially,
building blocks are randomly positioned in the binding
site. Subsequent free movement of the building blocks
is guided by molecular dynamics equilibria. Every
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 REVIEWS

few molecular dynamics steps, one or more building Later approaches3639 exclusively regarded interactions
blocks are randomly chosen, all bonds are cleaved and between the building blocks and the receptor (consen-
empty valences are filled with bonds to nearby building sus molecular dynamics). A further addition to this
blocks. The first such program, CONCERTS35, consid- method of structure sampling was a repeated cycle of
ered interactions between the building blocks and the construction and deconstruction: after a certain period
receptor, as well as between different building blocks. of molecular dynamics simulation, all candidate com-
pounds are dissected into their respective fragments.
Initial state Before each deconstruction, high-scoring structures
HO
O are stored in a list for later inspection38.
Ligand-based de novo design is not provided with
N NH O
interaction sites as primary target constraints. The
OH majority of ligand-based methods operate on the topo-
HN O logical molecular graphs and feature an evolutionary
algorithm for optimization. This choice implicitly paves
O the way for their structure sampling technique: genetic
O operators that are specifically tailored for molecular
O HN O graphs. Whereas Globus49 only implemented a recom-
+
N N bination operator, TOPAS48 solely applies a mutation
O N operator that randomly substitutes whole fragments,
OH
thereby obeying the rules of virtual chemical reaction
O 0.47
schemes. Nachbar45 and Brown50 developed both muta-
HO
O
tion and recombination operators. Candidate com-
pounds are mutated by changing an atom element type
O
or bond order, opening or closing rings and expand-
ing or contracting rings. Chemical Genesis22 applies
O genetic operators to the three-dimensional molecular
Tanimoto index (similarity to the template structure)

0.57 structures that led to the development of translational

and rotational mutation operators. BREED62, a recent
N
H
N NH
contribution to the field of de novo design, utilizes a
N
N N
completely different idea: a set of known ligands for
N N a particular target in their three-dimensional active
N O conformation is exhaustively recombined. The recom-
0.66 bination is carried out by overlaying the known ligands
O and swapping the fragments of different ligands on
each side of overlapping bonds. This procedure is car-
N NH N ried out recursively, so that the candidate compounds
that emerge from recombination are added to the pool
Br NH
of known actives and participate in subsequent cycles
of recombination. A prerequisite for BREED is knowl-
0.70
O edge of the receptor-relevant, presumed active ligand
H
N N conformation. As the known ligands are superimposed
N N NH N in their active conformation, the generated structures
N inherit this active conformation and are effectively
N Br NH
pre-docked to the receptor.
O
OH 0.81
Figure 4 | Progress of a de novo design exercise
HN following the concept of the design software TOPAS48
N N for assembling drug-like structures. The task was to
F N HN (re)generate the structure of imatinib (Gleevec; Novartis), an
O inhibitor of Abelson tyrosine kinase94. The algorithm started
off with a population of 100 randomly assembled molecules
O N 0.92 and navigated through the search space by stochastic
sampling. For molecule assembly, building blocks were used
N N N N that originated from pseudo-retrosynthetic fragmentation of a
H H
N database of drug-like molecules. Only the best partial
N F
solutions of selected generations are shown. The Tanimoto
index, which is based on Daylight Fingerprints, served as the
O N fitness function. Larger values indicate a higher degree of
1.0
similarity between the intermediate solutions and the
N N N N template structure, imatinib. After 50 generations, the
H H
N algorithm successfully reproduced the template. Individual
N End state
runs differed in terms of the highest-scoring candidate.

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2005 Nature Publishing Group
REVIEWS

O
OH
HO O H O N
O N N N
S H
N
O O H
O H2N S
N
OH

1 2 3

N O
HN F3C
HN
O CF3

O
4 5

Figure 5 | Examples of pharmacologically active substances that were designed de novo using computer algorithms.

Combinatorial search strategies paths reaches an end state. During a breadth-first

NPHARD
Non-deterministic
polynomial-time hard
(NP-hard) refers to a class of
decision problems of which
current knowledge provides
no way to obtain or derive a
solution time that is less than
exponential in problem size.
HEURISTIC
Application of probabilistic
rules grounded on knowledge
of a particular problem domain
to obtain an algorithm that
performs reasonably well in
many cases, but without proof
that it is always fast.
De novo design has to tackle the issue of combinatorial
explosion, which leads to problems that are NPHARD.
This class of problems is believed not to be solvable
with provably good run times and provably optimal
solution quality. Combinatorial search algorithms
offer a practical solution by giving up one or both of
these two aims. In other words, a combinatorial search
strategy usually finds pretty good solutions, but there is
no proof that the solutions will not become arbitrarily
bad; it usually runs reasonably quickly, but there is no
argument this will always be the case. A combinatorial
search algorithm is able to solve instances of combina-
torial problems by reducing the size of the search space
and by exploring it efficiently. HEURISTIC algorithms
represent one way to achieve this.
The search space for structure generation can be
represented by a graph in which each possible state of
the system is assigned a node (FIG. 3). The root node
represents the initial state (that is, the start of the
problem); terminal nodes of the graph correspond
to end states (that is, acceptable solutions of the
problem); and all other nodes represent intermediate
states. Application of a rule to a given node leads to
the generation of a successor node at the next level of
the graph. The objective of any search algorithm is to
find a path between the root node and the end nodes
by selecting the nodes that are expanded to the next
level of the graph.
Breadth-first and depth-first search
Several de novo design programs implement either a
breadth-first strategy23,2830,5759 or a depth-first strat-
egy15,18,28,3234,5759. The depth-first strategy retains only
one of a variety of possible partial solutions at each
level of the search space graph until an end state is
reached. Even if the highest-scoring partial solution
is selected each time, it is not guaranteed to find the
overall best solution, but the search space is reduced
significantly. A breadth-first strategy retains all partial
solutions at one level of the search space graph and
explores, sequentially, other levels until each of these
search all nodes are systematically examined so that
identifying the optimal solution is guaranteed.
How is such an exhaustive search in a de novo design
run possible? Most of the programs that implement the
breadth-first strategy deal with a smaller problem space:
they use the linking method for structure assembly.
Given that the key interaction sites are already satis-
fied with favourable ligand fragments, and a small set
of fragments is used to link them, an exhaustive search
is feasible. RASSE23, an atom-based growing approach
with a breadth-first search, implements another strategy.
It limits the search space by choosing only the 100 best
partial solutions at each growing step. An ancestor of
SPROUT32 and SPROUT itself5759 use the A* search
algorithm, a particular type of best-first search. The A*
algorithm optimizes a depth-first search by estimat-
ing the costs of reaching a specific partial solution and
reaching an end state from this partial solution. The
cost estimation is based on a heuristic that incorporates
knowledge about the problem domain in otherwise
general search algorithms. A refined version of the
combinatorial search algorithm of SPROUT led to an
interesting amalgamation of a breadth-first and a depth-
first search17. Another application of depth-first search is
the random selection of one partial solution at each tree
level among the highest-scoring partial solutions18,34 or
among all partial solutions40.
Monte Carlo and the Metropolis criterion
Pure random sampling, also called Monte Carlo
search, is implemented in the program LEGEND20.
Random sampling can also be combined with a
Metropolis criterion. In this case, after each struc-
ture-modification step, the change is evaluated
to decide whether it is accepted or rejected. If the
modification results in a fitter candidate compound,
it is immediately accepted. If, on the other hand, the
modification yields a less fit candidate compound, it
can still be accepted with a probability that is based
on the scoring function difference between the
modified and unmodified structure and a random

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2005 Nature Publishing Group

W60D NH
Y60A
H57
O
L99
S195
NH NH2
6 mosomes. It encodes the molecular structures in the
N98
A190
G216
W215 G219
D189
Figure 6 | Experimentally determined binding mode of benzamidine within the S1
substrate-recognition pocket of thrombin (by X-ray, resolution 3.16 , PDB identifier:
1DWB). Residues flanking the binding pocket are shown, including the catalytic Ser195. The
arrow indicates the direction of fragment growing by de novo design. The binding pocket offers
several potential interaction points with the ligand.
number. CONCEPTS39 was the first de novo design
program that made use of a Monte Carlo search with
the Metropolis criterion; several others followed
suit21,26,35,41,42,46,61,63,64.
Evolutionary algorithms
Evolutionary algorithms are based on the ideas
described by Charles Darwin in 185965. They are
population-based optimization algorithms that mimic
biological evolution with the genetic operators repro-
duction, mutation and recombination (crossover). Several authors have faced this problem and developed
Mutation introduces new information into a popula-
tion, whereas recombination exploits the information
inherent in the population. Candidate compounds are
represented by individuals in a population so that a set
of solutions is obtained in a single run of the algorithm.
The scoring function, which in the context of evolu-
tionary algorithms is often called a fitness function,
assesses the fitness of the individuals and therefore
determines which structures are chosen as parents. A
parent structure is then modified by one of the genetic
operators and the resultant child structure is put in the
population of the next generation. This cyclic process
of variation and selection is iterated until a termination
criterion is reached (FIG. 4).
Evolutionary algorithms can be further subdi-
vided into genetic algorithms, genetic programming
and evolution strategies. Genetic algorithms encode
the phenotype (molecular structure) by means of a
chromosome, which usually comprises either binary
SECONDARY TARGET
CONSTRAINTS
or real values. In genetic programming, the chro-
Essential drug properties apart mosomes are represented as trees rather than the
from the binding affinity to a fixed-length strings of genetic algorithms. This tree
biological target for example, representation facilitates the extension and contrac-
absorption, distribution,
metabolism, excretion and
tion of chromosomes. Evolutionary strategies omit
toxicity properties, or binding the encoding of phenotypic features by a chromo-
selectivity. some and operate directly on the phenotype instead.
NATURE REVIEWS | DRUG DISCOVERY
2005 Nature Publishing Group
REVIEWS
In structure generation, the phenotype itself is a
molecular graph that renders a distinction between
an evolutionary strategy and a genetic program-
ming algorithm difficult. Moreover, extensions to
genetic algorithms allow the use of extensible and
contractible chromosomes that are similar to those in
genetic programming. We therefore do not attempt
to classify the following examples any further than as
evolutionary algorithms.
The program LEA47 uses a tailored form of chro-
form of SMILES strings66 and the genetic operators are
carried out on these strings. In TOPAS48, the search for
candidate compounds is guided by an evolutionary algo-
rithm that features adaptive step-size control. The vari-
ance distribution of offspring is determined by a special
parameter. This parameter is also subject to evolution
so that the width of the search-space sampling that is,
the chemical diversity of the population adapts to the
local requirements of the fitness landscape.
The crossover operator can give rise to problems if
applied to cyclic graphs. As the name suggests, a cyclic
graph contains one or more cycles, which translate
to chemical ring systems in the context of molecular
structures. Cycles are a common property of molecular
structures and abundant in drug molecules. A crossover
operator takes two molecular graphs as an input, dis-
connects each graph into two subgraphs, and merges
two subgraphs from different input graphs. This pro-
cedure results in two molecular graphs, each of which
comprises parts of the first and the second input graph.
In graphs with cycles, the removal of a single edge does
not necessarily lead to two disconnected fragments.
Cyclic graphs therefore significantly complicate matters.
different solutions by modifying the crossover operator
appropriately22,45,49,50,67. There are other programs that
mainly utilize the population-based variation and selec-
tion cycle of evolutionary algorithms without directly
relating to the genetic operators24,55.
Recent examples of de novo design
There exist several published examples of successful
de novo design projects, and comprehensive lists of
applications have been compiled elsewhere6870. Here,
we present selected examples of more recent de novo
design efforts. Our intention is to emphasize attempts
aiming at synthetic accessibility and the incorpora-
tion of SECONDARY TARGET CONSTRAINTS to increase the
drug-likeness of the designed structures (FIG. 5).
In a semi-automatic design exercise, MCSS 27
was used to explore key regions in the active sites
of Candida albicans and Mycobacterium tuberculosis
lanosterol 14-demethylase (CYP51), a member of the
cytochrome P450 superfamily. Through this approach,
energetically favourable positions and orientations of
functional groups were obtained71. LUDI13,14 fragment
placing helped further explore important regions of
the binding sites. Initial lead molecules were manu-
ally constructed from a selection of the MCSS minima
that took into account synthetic feasibility. Subsequent
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REVIEWS
O The structure-based SYNOPSIS55 approach was
Y60A W60D
N
N
O rithm produced a shortlist of 28 candidates predicted
F
3.1
N98 HN NH2
G219 7
W215
G216
A190
D189
Figure 7 | Experimentally determined binding mode of compound 7 within an X-ray
model of the thrombin active site (resolution 1.67 , PDB identifier: 1OYT). Amino-
acid residues interacting with the ligand are highlighted. An FHC hydrogen bond was
shown to significantly enhance the proteinligand interaction. The hydrogen bond pattern
between the benzamidine moiety of the inhibitor and protein side chains in the S1
recognition pocket is indicated by dotted lines.
energy minimization and LUDI scoring led to the
discovery of a novel series of antifungal agents (such
as compound 1 in FIG. 5) with micromolar activity.
Compound 1 specifically interacts with active-site
residues and therefore potentially would not cause
toxicity arising from coordination binding with
the haeme of mammalian P450s. The secondary
constraint of avoiding P450 toxicity was indirectly
considered by restricting the ligandreceptor inter-
action to only those functional groups that form
non-covalent bonds between the ligand and residues
of the binding site. This example shows that manual
intervention at the level of fragment linking can
facilitate the identification of drug-like agents.
Embarking on a strict fragment-based approach,
the design software BREED62 was used to produce
novel human immunodeficiency virus 1 (HIV-1)
protease and kinase inhibitors following a two-step
protocol. In the first step, four known ligands of
each class were processed by the BREED recombina-
tion algorithm, and in the second step the resulting
hybrids were fused with additional known reference
molecules, resulting in more than 100 potential new
ligands. Compound 2 (FIG. 5) represents an example
of such a designed, potent HIV-1 protease inhibitor
(Ki = 42 nM). Both synthetic feasibility and potential
drug-like properties of the designs were gained by
starting off from drug-like reference molecules. In a
strict sense, the resulting molecules do not represent
real de novo designed structures; rather, they could
be regarded as chimeric structures that were generated
from exhaustive recombination of existing ligands.
The BREED concept demonstrates the usefulness
of re-using already known, potentially preferred,
fragments. It is noteworthy that BREED recombined
experimentally determined ligand conformations.
660 | AUGUST 2005 | VOLUME 4
2005 Nature Publishing Group
Consideration of automatically docked molecules
would further extend the numbers of ideas generated
by this algorithm62,72.
used to find new inhibitors of the HIV-1 protein reverse
transcriptase (RT). Several runs of the design algo-
to have low IC50 values. The designs were assessed
by an organic chemist for both synthetic feasibility
and sufficient dissimilarity from known HIV-1 RT
inhibitors. Of 18 molecules synthesized and tested, 10
compounds were identified with inhibitory in vitro
activity below 100 M, four were cytotoxic molecules
and four were inactive. Compound 3 (FIG. 5) gives an
example of a designed HIV-1 RT inhibitor (IC50 =
4.4 M). The authors stress that the outcome of the
design project and hit rate compares favourably with
HTS experiments despite the fact that a rather high
activity threshold was used52,55,73.
LEGEND20 was applied to the design of new cyclin-
dependent kinase 4 (CDK4) inhibitors in a structure-
based manner from a homology model of CDK456. The
designs were not synthesized; instead, combinatorial
scaffolds with molecular masses below 350 Da were
extracted from the candidate molecules and served
as templates for library design. In a first step, a novel
class of CDK4 inhibitors was found with nanomolar
inhibitory activity (compound 4; the scaffold is shown
in red in FIG. 5). Subsequently, a combination of LUDI
and LeapFrog (Tripos Inc.) was used to design a
pyrazol-3-ylurea library of selective low-nanomolar
CDK4 inhibitors74. It is noteworthy that the design
algorithms were only used for the identification of
side chains of the combinatorial scaffold that show
favourable interactions with enzyme residues around
the ATP-binding pocket. These examples demonstrate
the potential of combining established chemistry that
ensures synthetic accessibility with de novo design for
side-chain optimization.
A related, entirely ligand-based, concept was
pursued using TOPAS48 for the design of combina-
torial libraries directed against human cannabinoid
receptor 1 (CB1)75. A stock of building blocks was
generated from pseudo-retrosynthetic fragmentation
of known GPCR ligands. These building blocks were
used for subsequent de novo design. A single known
CB1 ligand served as the reference for pharmacoph-
ore-based fitness calculations. From several TOPAS
runs, two prominent scaffolds were identified, and
small, focused libraries were devised by similarity
searching for promising and physically available side
chains. Hit rates of 6% and 10% were obtained, respec-
tively, applying an activity threshold of Ki = 10 M.
The most potent ligand was compound 5 (Ki = 0.3
M; scaffold shown in red in FIG. 5). This study dem-
onstrates that novel lead-structure candidates can be
found using ligand-based de novo design even in the
absence of receptor-structure information by taking
a single known reference ligand as a template. Again,
the combination of de novo design and combinato-
rial optimization was pivotal to success. The hit rate
www.nature.com/reviews/drugdisc

was up to two orders of magnitude higher than that
expected for random screening of historical corporate
compound collections. Lengthy assay development
and costly, logistically complex, HTS efforts were not
required in order to generate two novel, selective and
patentable hit series whose chemical simplicity can
serve as a starting point for further refinement and
lead-optimization activities.
How do such automated, entirely computer-gener-
ated designs compare with rational, non-automated
design approaches for example, by manual molec-
ular modelling, docking and piecemeal structure
modification? The design of non-peptidic thrombin
inhibitors has been the focus of many de novo
design projects during recent years and can help
answer this question.
Thrombin is a trypsin-like serine protease with a
central role in the blood-clotting cascade. A pivotal
part of current thrombin inhibitors is a functional
group interacting with Asp189 at the bottom of the
S1 recognition pocket of the enzyme. As thrombin
cleaves fibrinogen after an arginine residue, guanidin-
ium-mimicking fragments are usually selected by de
novo design software at this position. The best result
of an automated combinatorial docking and design
approach using LUDI is compound 6 (Ki = 10 nM),
which contains a benzamidine in this position (FIG. 6)76.
In this study, p-amino-benzamidine (Ki = 34 M) was
identified as a preferred core fragment for subsequent
combinatorial optimization by LUDI (unsubstituted
benzamidine alone yields a Ki of 250 M).
For comparison, a carefully, rationally de novo
designed molecule is compound 7 (FIG. 7), which was
optimized in a step-wise manner to display selective
thrombin inhibition77. Also revealing the benzami-
dine residue in the S1 pocket, this compound was
developed from a rigid tricyclic core structure that
pre-organizes this chemotype for thrombin bind-
ing78. As a result of a fluorine scan of the precursor
molecule, an FHC hydrogen bond was shown to De novo design offers a broader exploration of
significantly enhance the proteinligand interaction
(Ki = 6 nM; 67-fold selectivity compared with trypsin
inhibition) (FIG. 7). The increased positive polarization
of the hydrogen in the ortho position to the fluor sub-
stituent is also supposed to enhance the edge-to-face
interaction with Trp215. Such effects and other types
of ligandreceptor interactions, such as arenearene
and cation interactions, or water-mediated con-
tacts, are often neglected by de novo design software.
These non-covalent interactions can influence the
binding mode and affinity of a ligand, and therefore
demonstrate limitations of current automated de novo
design methods. Starting the amalgamation of frag-
ments from small seed structures, such as the ben-
zamidine fragment in the thrombin example, might
even prevent better solutions from being found.
Conclusions
The drug discovery pipelines of many pharmaceu-
tical companies are fuelled by HTS as one of the
major sources of new hit-to-lead candidates. This is
NATURE REVIEWS | DRUG DISCOVERY
2005 Nature Publishing Group
REVIEWS
a direct consequence of our lack of knowledge about
function-determining features of desired ligand mol-
ecules in the early stages of the discovery process79.
We expect de novo design to increasingly become a
complementary strategy to HTS through a number of
routes: by suggesting new chemical entities that were
designed taking into consideration several aspects of
lead- and drug-likeness and synthetic accessibility;
by making full use of pre-existing knowledge, such
as reference ligands or receptor models; and by facili-
tating the design of activity-enriched screening sets
with increased hit rates.
Automated de novo design has proven its value
for hit and lead-structure identification. Designed
molecules provide sometimes astonishing ideas for
the medicinal chemist and aid in the development
of novel and patentable leads with desired property
profiles. To gain further acceptance of computer-
generated molecules it will be essential to understand
current limitations of the design techniques. One
weakness is the inability of de novo design software
to sufficiently consider the flexibility of the target
protein. Although examples of docking methods exist
that consider receptor flexibility, examples in the field
of de novo design are sparse38,80. This is probably a
result of the combinatorial problem de novo design
is faced with; receptor flexibility adds just another
aspect of combinatorics on top of this problem. The
same holds for flexible alignments of ligand ensembles
that provide the basis for a QSAR, pharmacophore
or pseudo-receptor model81. One cannot expect good
designs from poor initial alignments, because these
guide the search through chemical space.
Most pharmaceutical leads are part of a limited set
of chemotypes8284. Many pharmaceutical companies
direct their attention towards this limited number of
structural classes. The chemical diversity of potential
leads discovered by HTS is therefore restricted by
the diversity of the screening libraries that are used.
chemical space and therefore makes it possible to
identify novel ligand scaffolds, which can be a major
competitive advantage85. Fragment-based screening
strategies, such as NMR and high-throughput X-ray
crystallography, can be used to help identify new
chemotypes by suggesting starting orientations of
molecular building blocks for subsequent computer-
assisted linking and growing86,87. Still, the prediction
of crucial properties of a drug molecule primary
target constraints (binding behaviour) as well as
secondary constraints such as pharmacokinetic
properties is limited. Molecules that are predicted
to be the best from a de novo design run rarely rep-
resent the preferred choice of a medicinal chemist.
Even though there is fully automated de novo design
software, it remains a crucial human task to pick the
most promising candidates. An important goal of de
novo design is to inspire medicinal chemists through
the chemical motifs that are identified. Ultimately, the
aim is to offer support for hit and lead identification
and widen the chemical horizon.
VOLUME 4 | AUGUST 2005 | 661
REVIEWS
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Acknowledgements
H. Kubinyi is thanked for helpful discussion and kind support. This
work was supported by the Beilstein-Institut zur Frderung der
Chemischen Wissenschaften, Frankfurt am Main. U.F. is thankful
for a fellowship granted by Aventis Pharma Deutschland GmbH,
a company of the Sanofi-Aventis group.
Competing interests statement
The authors declare no competing financial interests.
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