J. Mol. Biol.

(1995) 245, 43-53

Molecular Recognition of Receptor Sites using a
Genetic Algorithm with a Description of Desolvation
Gareth Jones!, Peter Willett and Robert C. Glen**
1Dep7rtnwr~t of lt@m7tior7
Studies rind Krebs lnsfittife
for Biomolecular Resenrclz
University of Sheffield
Western Bank, Sheffield SlO
2TN, U.K.
Deprtment of Plzysicnl
Sciences, Wellcome Resenrh
Laboratories, Beckenham, Kent
BR3 385, U.K.
*Currcsyondirrg niffhor
Understanding the principles whereby macromolecular biological receptors
can recognise small molecule substrates or inhibitors is the subject of a major
effort. This is of paramount importance in rational drug design where the
receptor structure is known (the docking problem). Current theoretical
approaches utilise models of the steric and electrostatic interaction of bound
ligands and recently conformational flexibility has been incorporated. We
report results based on software using a genetic algorithm that uses an
evolutionary strategy in exploring the full conformational flexibility of the
ligand with partial flexibility of the protein, and which satisfies the
fundamental requirement that the ligand must displace loosely bound water
on binding. Results are reported on five test systems showing excellent
agreement with experimental data. The design of the algorithm offers insight
into the molecular recognition mechanism.
E(etJruor&s: protein ligand docking; genetic algorithm; desolvation
Introduction
Biological receptors exhibit highly selective
recognition of small organic molecules. Evolution
has equipped them with a complex three-dimen-
sional lock into which only specific keys will fit.
This has been exploited by medicinal chemists in
the design of molecules to selectively augment or
retard biochemical pathways and so exhibit a clinical
effect. X-ray crystallography has revealed the
structure of a significant number of these receptors
or active sites. It would be advantageous in
attempting the computer-aided design of therapeutic
molecules to be able to predict and explain the
binding mode of novel chemical entities (the
docking problem) when the active site geometry is
known.
The process of automated ligand docking has
been the subject of a major effort in the field
of computer-aided molecular design (Blaney &
Dixon, 1993). Early examples, for example the
DOCK program (Kuntz et al., 1982), consider only
orientational degrees of freedom, treating both
ligand and receptor as rigid (DesJarlais et al.,
1988; Shoichet et al., 1993; Lawrence & Davis,
1992).
Abbreviations used: GA, genetic algorithm; NMR,
nuclear magnetic resonance; DHFR, dihydrofolate
reductase; NADPH, reduced nicotinamide adenine
dinucleotide phosphate; r.m.s., root-mean square;
4g-neu5Ac2en,4-guanidino-2-deoxy-2,3-di-dehydro-o-N-
acetylneuraminic acid.
0022-2836/95/010043-11 $08.00/O
More recently algorithms that take into account
the conformational degrees of freedom of the
ligand have been reported. These include docking
fragments of the ligand and recombining the
molecule (DesJarlais et al., 1986), systematic search
(Kuntz, 1992) and clique detection (Smellie et al.,
1991). These algorithms are all inherently combina-
torial. Another approach is simulated annealing
(Goodsell & Olson, 1990). Unfortunately the
algorithm is very time-consuming, even given
the relatively simple molecules docked. Dixon
(1993) has given a brief report on the use of a
genetic algorithm (GA) for docking, however,
experimental results are as yet unpublished. Leach
(1994) has developed an algorithm that takes
into account conformational flexibility of protein
side-chains as well as ligand flexibility; These
results highlight the need for appropriate methods
to accurately estimate the strength of ligandsbinding.
A solution to the docking problem requires a
powerful algorithm to search conformational space
and an understanding of the processes of molecular
recognition, so that reliable predictions of binding
modes can be achieved.
Inspection of the X-ray crystallographic structures
of proteins with associated higli affinity Iigands
reveals that they appear to conform closely to the
shape of the receptor cavity and to interact at a
number of hydrogen-bonding sites. The docking
process probably involves the exploration of an
ensemble of binding modes with the most
energetically stable being observed. These exhibit
low temperature factors and little crystallographic
0 1995 Academic Press Limited
44
Molecular Recognition of Receptor Sites
disorder,The key requirements for high affinity may
be the complementarity of the three-dimensional
shape of the ligand and protein, and the ability of the
ligand to displace water and form hydrogen bonds al-
key hydrogen-bonding sites when associated with
the active site. Thus sufficiently accurate simulation
of these interactions may be enough to explain and
predict the binding mode of the majority of
high-affinity ligands. /
Account must be taken of the ligand and protein
conformational variability Here, we have adopted a
genetic algorithm (Davis, 1991; Goldberg, 1989;
Holland, 1975) that uses an evolutionary strategy in
exploring the full conformational flexibility of the
ligand with partial flexibility of the protein. GAS are
a class of computational procedures that enable the
rapid identification of good, but not necessarily
optimal, solutions to combinatorial optimisation
problems. There have been many reports of the use
of GAS for a range of combinatorial matching
problems in chemistry and biology (Brown et ai.,
1994; Fountain, 1992). In particular, GAS have shown
success in the conformational analysis of both small
molecules (Clark et nl., 1994; Payne & Glen, 1993;
Judson ef nl., 1993) and of macromolecules
(Blommers et nl., 1992; Dandekar & Argos, 1993).
Tlie GA to be described here performs automated
docking with full acyclic ligand flexibility and partial
protein flexibility in the region of the receptor or
active-site. To enable the GA to explore the possible
hydrogen-bonding motifs we have attempted to
quantify the ability of common substructures of drug
molecules to displace water from the receptor
(protein) surface. This, in conjunction with a steric
.term, allows the GA to find the most energetically
favourable combination of interactions. The algor-
ithm has been tested on a number of ligand/protein
docking problems and five examples are reported
here.
Results and Discussion
The GA described in Materials and Methods
requires as input the location and the size of the
receptor site and the ligand that is to be docked into
this site. The output is the ligand and protein
conformations associated with the fittest chromo-
some in the population when the termination
conditions of the algorithm were satisfied. To ensure
that most of the high-affinity binding modes were
explored the GA was run 50 times for each of the
examples examined (probably less runs are required
as the top 20 results in most cases were very similar).
The result with the best fitness score was used as the
solution (although other results, with poorer fitness
scores, may have better fits to the crystal structures).
On average, each run of the GA took approximately
five to eight minutes of CPU time on a Silicon
Graphics R4000 Iris computer. Due to the inherently
parallel nature of the algorithm and the .recent
progress in CPU technology, the computing time may
be expected to be substantially reduced on more
recent processors.
Figure 1. Docking of methotrexate into ciihycirofolate
reciuctase.
The GA docking procedure has been applied to a
diverse set of problems, illustrated by Figures 1 to 5.
In these Figures, ligand crystal structures (where
available) and important protein close contacts have
been coloured by atom type. For reasons of clarity,
not all close contacts with the protein are displayed.
The GA solutions are shown in red.
Methotrexate and dihydrofolate reductase
Dihydrofolate reductase (DHFR) is an enzyme of
vital importance to DNA synthesis and preferential
inhibition of the enzyme has been exploited to
produce a range of potent anti-bacterial drugs and
anti-cancer drugs. The geometry of binding of the
anti-cancer agent methotrexate with Escl~ickin coli
DHFR has been elucidated by X-ray crystallography
(Bolin et nl., 1982). This is the binary complex (no
NADPH).
Bound methotrexate is protonated at N-l and
the binding mode of the pteridine ring results in
a number of close contacts: pteridine N-l
and 2-amino . . . Asp27 carboxylate, 2-amino
group . . . water, 4-amino group . . . Ile5 and he94
carbonyl groups, benzoylcarbonyl . . . Arg52 guani-
dinium (and water), L-glutamate cr-carboxy-
late . . . Arg57, L-glutamate y-carboxylate . . . Arg52
and various water molecules. Not all of these are
shown, for reasons of clarity
The GA solution (red) is shown overlaid upon the
experimental result (coloured by atom type) in
Figure 1.
Key hydrogen-bond interactions are shown in
yellow. The similarity between the predicted and
experimental results is excellent, with a root-mean-
square (r.m.s.) deviation for the atoms of 0.99 A.
The pteridine ring is correctly oriented with
the hydrogen-bond (pteridine N-l and 2-
amino . . . Asp27) the main feature. The benzoylcar-
bony1 group is correctly oriented, interacting with
Arg52. The L-glutamate y-carboxylate group is
rotated slightly from the X-ray solution (where it is
solvated), but the GA twists the side-chain to interact
with Arg52. The L-glutamate a-carboxylate group is
correctly positioned. The absence of bound water
Molecular Recognition of Receptor Sites 45
Figure 3. Docking of o-galactose into an L-arabinose
binding protein mutant.
from the GA model seems not to affect the ability of
the GA to correctly predict the binding mode.
Perhaps bound water is a consequence of binding
rather than a contributory factor to the stabilisation
of the bound ligand.
Folate and dihydrofolate reductase
Vertebrate DHFR catalyses the NADPH-linked
reduction of folate to 7&dihydrofolate and on to
5,6,7&tetrahydrofolate. The structure of the human
recombinant enzyme has been determined by X-ray
diffraction (Davis et al., 1990). At physiological pH,
folate appears to bind as the neutral species and
displays a number of close contacts in the active site:
pteridine ring 2-amino . . . Glu30, pteridine ring
2-amino . . . water . . . Thr136, pteridine ring
N-3 . . . Glu30, the pteridine ring carbonyl
O-4 . . . water . . . Glu30, the carbonyl group of the
y-aminobenzoic acid . . . Asn64, the folate a-car-
boxylate group of the glutamate . . . Arg70, the folate
y-carboxylate group of the glutamate. . . water
molecules (not all shown, for reasons of clarity).
The GA solution is shown in red with the X-ray
data coloured by atom type.
The GA correctly predicts the binding mode of the
pteridine ring with the pteridine ring distances
2-amino. . . Glu30 and the pteridine ring N-3
Glu30 similar to the X-ray result, with an r.m.s.
heiiation for the atoms in the pteridine ring of 0.63 A.
The benzene ring is rotated relative to the crystal
structure due the interaction of the folate glutamate
y-carboxylate group with Arg70. Solutions having
the alternative interaction (the folate cr-carboxylate
group of the glutamate . . . Arg70) were found by the
GA, however, the illustrated solution had the best
GA score with an r.m.s. deviation for the atoms in the
complete structure of 2.48 A. The X-ray result has one
carboxylic acid molecule in solvent while the GA
solution has both interacting with the protein.
Folate binding to DHFR has been studied by NMR
spectroscopy These studies have suggested that
three forms exist in equilibrium (Cheung ef nl., 1993).
The predominant form at low pH (form 1: enol, N-l
protonated) appears to bind in a similar manner to
methotrexate while the neutral form (form-2: keto
form with N-l unprotonated) binds with the
pteridine ring rotated 180. Also, a third protonated
form (form 3: enol N-l protonated) coexists with
form 2 (the protonation state of folate in the protein
in forms 2 and 3 is relatively insensitive to buffer pH)
and this also binds in a similar mode to methotrexate.
The GA solution for form 2 has previously been
described. Form 3 is predicted by the GA to bind
with the pteridine ring rotated by 180; this
prediction is shown in yellow in the Figure. This is
consistent with the NMR results.
In the X-ray experiment (crystallisation performed
at pH 5.91, difference maps were used to determine
the folate position. However, extra electron density
near the pteridine and benzene rings could not be
accounted for. It may be speculated that the X-ray
experiment observes forms 2 and 3 in equilibrium
and the two GA binding results in combination
would then account for the extra density
Galactose and L-arabinose binding protein
The structure of the carbohydrate-binding protein,
L-arabinose binding protein mutant (Metl08Leu) has
been determined by X-ray crystallography (Vermer-
sch et al., 1991). This is a mutant of a periplasmic
transport receptor of E. coli with high affinity for the
sugar L-arabinose. The ligand (D-galactose) position
was determined from difference Fourier analysis.
There are many close contacts with the protein:
o-1 . . . Asp90, O-2 . . . water, o-3 . . . Glu14,
o-4 . . . Asn232, LyslO . . . O-2,
Asn232 . . . O-3, ArglSl . . . O-4,
Asn295 . . . O-3,
Argl51 . . . O-5,
water . . . O-5 (not all of these are shown, for reasons
of clarity).
The GA solution (Figure 3) is almost identical with
the experimental result, with the r,m.s. deviation of
the atoms being as low as 0.67 A with all of the
experimentally observed close contacts being cor-
rectly reproduced.
Again, the GA predicts the correct binding mode
despite the absence of bound water in the model.
Ring flexibility of the ligand was not included in
the GA run. This makes the problem somewhat
easier, but such flexibility could be included, if
required, by the incorporation of a corner-flapping
46
Molecular Recognition of Receptor Sites
Figure 4. Docking of 4-guanidino-2-deoxy-2,3-di-dehy-
dro-D-N-acetylneurantic acid into influenza sialidase.
Figure 5. Docking of trimethoprim into DHFR (EC
1.5.1.3.) complex with NADPH.
algorithm (Payne &Glen, 1993). The conformation of
the l&and used in the GA is consistent with the
known stable conformations of sugar molecules in
solution. The alternative epimer (L-galactose, results
not shown in diagram for clarity) was also docked by
the GA. The solution that was obtained was again
very close to the experimental result, with an r.m.s.
deviation of 0.66 A.
4-Guanidino-2-deoxy-2,3-di-dehydro-D-N-
acetylneuraminic acid and influenza sialidase
Potent inhibitors of influenza virus replication
have recently been reported based upon inhibition of
influenza sialidase. The binding mode of the
inhibitor 4-guanidino-2-deoxy-2,3-di-dehydro-D-N-
acetylneuraminic acid (4g-neu5Ac2en) to sialidase
was reported based upon X-ray crystallography
analysis of the protein with bound ligand (von
Itzstein et nl., 1993). The ligand binds with a number
of reported close contacts: terminal guanidinyl
nitrogen atoms . . . Glu227 (2.7 A), terminal
guanidinyl nitrogen atoms. . . Glu119 (3.6 and
4.2 A), carboxylate . . . Arg371.
The GA solution (red) is overlaid on the crystal
structure of sialidase (coloured by atom type) in
Figure 4.
This solution has been compared with the
reported intermolecular distances, difference map
and diagrams (von Itzstein et nl., 1993). It appears
from these that the GA correctly predicts the binding
mode, closely mirroring the experimental result. The
GA solution is shown in red with close intermolecu-
lar contacts in yellow. In addition the GA identifies
a strong contact with Aspl51.
Trimethoprim and dihydrofolate reductase
Complexes of DHFR and trimethoprim (5-(3,4,5-
trimethoxyphenyl)methyl - 2,4 - pyrimidinediamine)
have been solved for a number of different DHFR
species. Trimethoprim has been seen to bind in at
least two different forms, the E. coli mode (form 1;
Matthews et al., 1985) or the avian mode (form 2;
Matthews et al., 1985). The diaminopyrimidine stays
largely in the same position in both forms, however,
the trimethoxybenzene has rotated due to altered
torsional angles about the methylene ring-linking
group.
The nature of the active site is such that the E. coli
site is more restricted than the mammalian sites. This
is reflected in the single binding mode observed for
trimethoprim/E. coli DHFR while different modes of
binding of trimethoprim are observed in mammalian
DHFR types.
In mouse L1210 (Stammers, 1987) it is bound as
form 2. With mouse S180 after co-crysfdlisnfion of
trimethoprim with the DHFR, it binds as form 1
(Groome, 1991). With mouse S180 after diffikorz of
trimethoprim into the DHFR, it binds in form 2 (A.
J. Geddes, personal communication).
We have applied the GA to both the E. co/i and
mammalian forms.
The ternary complex of DHFR (EC 1.5.1.3),
NADPH and trimethoprim (Champness ef nl., 1986)
was partially crystallographically refined (J. N.
Champness, personal communication). The
trimethoprim molecule interacts via the diaminopy-
rimidine with Asp27 forming a pair of hydrogen
bonds. The implication is that N-l of trimethoprim is
protonated. One methoxy group is positioned in van
der Waals contact with the side-chains of Leu28 and
Phe31.
The genetic algorithm solution (red) is overlaid on
the crystal structure (Figure 5). It is close to the
crystallograpl$ally determined structure (r.m.s.
deviation 1.1 A) having an almost identical confor-
mation and being slightly displaced. Of particular
note is the positioning of the trimethoxybenzene
moiety very close to the crystallographically
determined structure.
The ternary complex of mouse L1210 DHFR/
NADPH and trimethoprim (Stammers et al., 1987)
was used as a representative of a marnmalian DHFR.
In this crystal structure, the trimethoprim is bound
in a conformation consistent with form 2.
As described, the GA was run 50 times and the
solutions ranked according to their fitness scores. In
Figure 6, the first six GA runs (green) corresponded
closely to form 1 while it is not until the tenth GA run
Molecular Recognition of Receptor Sites
47
Figure 6. Docking of trimethoprim into mouse L1210
DHFR. The experimental solution is coloured by atom
type. The highest scoring GA solutions (runs 1 to 6) are
typified by the green molecule (close to the bacterial mode
of binding, (for pictures of this site, see Groome et nl., 1991))
while the closest GA solution (10th best score) to the
experimental solution is coloured in yellow.
(yellow) that solutions close to the observed form 2
are seen.
Whilst it might have been more satisfying if form
2 (the experimental result in this study) had
appeared more favourable in terms of the GA score,
these results are nevertheless a reflection of the
relatively low affinity of trimethoprim for the
mammalian receptor coupled with the possibility of
multiple binding modes. The two binding modes
observed are probably very close in relative affinity
for this receptor. A simple change such as
crystallisation conditions results in conformational
changes in the observed binding conformation of
trimethoprim. Indeed, the GA results imply that
other modes of binding of the trimethoxybenzene
portion of the molecule may be observed under
different crystallisation conditions. This conclusion
is corroborated by the crystallographic observations
of trimethoprim binding to L1210 DHFR (J. N.
Champness et nl., 1994).
Reproducibility
Since the genetic algorithm is stochastic in nature,
there may be problems with reproducibility and
convergence. In our experience with the examples
quoted here, the docking experiments using
galactose, methotrexate, trimethoprim (E. co/i) and
4gneu5Ac2en were highly reproducible. Folate and
mammalian DHFR presented a much more difficult
problem and required many GA runs (typically 20 to
30) to elucidate reliably the observed (crystallo-
graphic) binding mode.
Conclusions
Here, we have described the use of a GA to dock
flexible ligands into protein binding sites. The results
that have been described in the previous section and
that are illustrated in Figures 1 to 6 demonstrate the
effectiveness of the algorithm in reproducing
crystallographic studies of the bound complexes,
even in the case of highly flexible ligands with
variable protonation states. The accuracy with which
the binding modes of ligands are predicted suggests
that the representation and fitness function used here
provides a useful description of the most important
features involved in binding, at least for these
high-affinity ligands. If this is indeed so, our results
would suggest that the mechanism of molecular
recognition requires the ligand to exhibit shape
complementarity with the receptor (as has been
assumed by previous docking programs) and to
displace water at key points so forming hydrogen
bonds in preference to water. The absence of bound
water from the GA model (all water molecules are
removed from the protein before docking) seems not
to affect the ability of the GA to correctly predict the
binding mode. Perhaps the bound water (in these
examples) is a consequence of binding rather than a
major contributory factor to the stabilisation of the
bound ligand.
Several improvements to the algorithm are
possible (such as ring conformational search, the
inclusion of metal ions and accounting for possible
variations in dielectric and solvent competition
across the active site) and subsequent developments
will be published elsewhere. Also, the dependence
of this implementation on mapping hydrogen bonds
from ligand to protein may not reflect the situation
found in predominantly hydrophobic ligands. We are
currently evaluating a number of alternative
intermolecular potential functions (we currently use
a simple Lennard-Jones 6-12 potential) to describe
better the hydrophobic interactions and the results
will be reported elsewhere.
Materials and Methods
Genetic algorithms
A GA is a computer program that mimics the process of
evolution by manipulating a collection of data-structures
called chromosomes. Each of these encodes a possible
solution (in terms of a possible ligand-receptor interaction)
to the docking problem and may be ass_igned a fitness score
based on the relative merit of that solution. A
steady-state-with-no-duplicates GA (Davis, 1991) with a
fixed population size of 500 was used. An overview of this
GA is given in Figure 7.
Starting from an initial, randomly generated population
of chromosomes the GA repeatedly applies two genetic
operators, crossover and mutation resulting in chromo-
somes that replace the least-fit members of the population.
Crossover combines chromosomes while mutation intro-
duces random perturbations. Both operators require
parent chromosomes that are randomly selected from
the existing population with a bias towards the fittest, thus
introducing an evolutionary pressure into the algorithm.
This selection is known as roulette-wheel-selection, as the
procedure is analogous to spinning a roulette wheel with
each member of the population having a slice of the wheel
that is proportional to its fitness. This emphasis on the
survival of the fittest ensures that, over time, the
population should move towards the optimum solution,
i.e. to the correct binding mode in the present application,
48
Molecular Recognition of Receptor Sites
1. A set of.reproduction operators (crossover, mutation etc) is chosen.
l&h operator is assigned a weight.
2. An initial population is randomly created and the fltnesses of its
members determined.
3. An operator is chosen using roulette wheel selection based on
operator weights.
4. The parents required by the operator are chosen using roulette wheel
selection based on scaled fitness. I
5. The operator Is applied and child chromosomes produced. Their
fitness Is evaluated.
6. If not already present hi the population. the children replace the least
fit members of the population.
7. If an acceptable solution has been found stop otherwise goto 3.
Figure 7. Steady state with no duplicates GA.
provided that the fitness function is an accurate predictor
of binding affinity.
The GA utilises a novel representation of the docking
process. Each chromosome encodes an internal confor-
mation of the &and and protein active site, and includes
a mapping from hydrogen-bonding sites in the ligand to
hydrogen-bonding sites in the protein. On decoding a
chromosome, a least-squares fitting process is employed to
position the ligand within the active site of the protein in
such a way that as many of the hydrogen bonds suggested
by the mapping are formed. The fitness of a decoded
chromosome is then a combination of the number and
strength of the hydrogen bonds that have been formed in
this way and of the van der Waals energy of the bound
complex. The remainder of this section gives a detailed
account of the various components of our program, these
being: the routines that are used to initialise the protein site
and the ligand that is to be docked into it; the chromosome
representation that is used to describe ligand and protein
conformations and the hydrogen bonds that are formed
between them; the fitness function that is used to evaluate
these chromosomes; and the genetic operators that are
applied to these chromosomes. We also discuss the
calculation of the hydrogen-bond energies that play a vital
role in the fitness function.
lnitialisation of the protein and of the ligand
Known crystal structures of bound ligand complexes
were obtained from the Brookhaven Protein Data Bank
(Bernstein et nl., 1977; BoLin et nl., 1982; Davis et nl., 1990;
Vermersch ef nl., 1991; von Itzstein et nl., 1993; Champness
ef nl., 1986). Water molecules and ions were removed
(including ordered water molecules) and hydrogen atoms
added at appropriate geometry Groups within the protein
(and ligand) were ionised if this was appropriate at
physiological pH. The structures were partially optimised,
Table 1
Allowed donors and acceptors based on SYBYL atom types
SYBYL atom types Donor Acceptor
N.3, N.2, 0.3 Y Y
N.l, N.ar, 0.2, 0.~02, F, Br, Cl N i-
N.am, N.pl3, N.4 Y N
N.B. Donors must have a hydrogen atom attached.
to relieve any bad steric contacts introduced by hydrogen
addition for ten cycles of molecular mechanics using the
SYBYL general purpose Tripos 5.2 force field (Clark cl al.,
1989). No further optimisation was employed so as not to
risk distorting the crystal structure. The l&and was
extracted from the structure and then fully optimised in an
extended conformation (in vnc~o) by molecular mechanics.
The remaining structure then comprised the protein active
site.
The analysis of the protein commenced with the use of
a flood-fill procedure to determine solvent-accessible
protein atoms (Ho & Marshall, 1990) within the active site.
Accessible hydrogen-bond donor and acceptor atoms were
identified using the SYBYL atom-type categorisation
(Clark et nl., 1989) in Table 1.
Lone pairs were added to acceptors at a distance of 1.0 A
(taking into account the hybridisation state of the atom).
Any single bond that connected a terminal donor or
acceptor to the protein was selected as rotatable. This
choice of rotatable bonds within the protein allows donors
and acceptors in the active site to rotate their lone pairs and
hydrogen atoms into the best position to form hydrogen
bonds with the ligand. This of course may be easily
extended to include side-chain flexibility (at greater
computational cost); however, at present only the potential
hydrogen-bonding groups have partial flexibility.
Hydrogen-bond donors and acceptors in the &and were
identified. Lone pairs were added as before. All acyclic
single non-terminal bonds were marked as rotatable.
Prior to docking a random translation was applied to the
ligand and random rotations were applied to all bonds
marked rotatable in the ligand and protein.
The chromosome representation
Each chromosome in the GA comprised four strings, two
of which were binary and two of which were integer in
character. Conformational information was encoded by
two gray-coded (Goldberg, 1989) binary strings: one for the
protein and one for the ligand, where each byte in the string
encoded an angle of rotation about a rotatable bond. Two
integer strings encoded mappings, suggesting possible
hydrogen bonds between the ligand and the protein active
site. The first integer string encoded a mapping from lone
pairs in the ligand to hydrogen atoms in the protein, such
that if V was the integer value at position P on the string,
then the chromosome mapped the Pth lone pair in the
ligand to the Vth hydrogen atom in the protein active site.
In a similar manner, the second integer string encoded a
mapping from hydrogen atoms in the ligand to lone pairs
in the protein. By associating hydrogen atoms with lone
pairs these mappings suggested hydrogen bonds between
the ligand and protein. On decoding the chromosome the
GA used a least-squares routine to attempt to form as many
of these hydrogen bonds as possible.
The fitness function
The fitness function was evaluated in six stages as
follows. (1) A conformation of the liga!ld and protein active
site was generated. (2) The ligand was placed within the
active site of the protein using a least-squares fitting
procedure. (3) A hydrogen-bonding energy, H-Bad-En-
crgy, was obtained for the complex. (4) A van der Waals
energy, Coqkx-VD W-Encrg~, was obtained for the
energy of interaction between the ligand and the protein.
(5) A van der Waals energy, lllterrlnl_VDW_Energy, was
Molecular Recognition of Receptor Sites
49
Table 2
Hydrogen-bonding energies
Energy (kcal/moU
Donor Acceptor Donor Acceptor Complex Bond
N4
N4
N4
N4
N4
N4
N4
N4
N4
N4
N4
N4
NPW
NPL3
NPW
NFL3
NPW
NPW
NPL3
NFL3
NW3
NPW
NPW
NPL3
NSDA
N3DA
N3DA
N3DA
N3DA
N3DA
N3DA
N3DA
N3DA
N3DA
N3DA
N3DA
NAM
NAM
NAM
NAM
NAM
NAM
NAM
NAM
NAM
NAM
NAM
NAM
03DA
03DA
03DA
03DA
03DA
03DA
03DA
03DA
03DA
03DA
03DA
03DA
NZDA
NZDA
NZDA
NZDA
NZDA
NZDA
N2DA
N2DA
NZDA
BR
N2DA
02
oco2
CL
Nl
N3A
03A
F
N2A
N3DA
03DA
BR
NZDA
02
oco2
CL
Nl
N3A
03A
F
N2A
N3DA
03DA
BR
NZDA
02
oco2
CL
Nl
N3A
03A
F
N2A
N3DA
03DA
BR
N2DA
02
oco2
CL
Nl
N3A
03A
F
N2A
N3DA
03DA
BR
N2DA
02
oco2
CL
Nl
N3A
03A
F
N2A
N3DA
03DA
BR
N2DA
02
oco2
CL
Nl
N3A
03A
F
- 24.784
- 24.784
- 24.784
- 24.784
- 24.784
- 24.784
- 24.784
- 24.784
- 24.784
- 24.784
- 24.784
- 24.784
- 4.427
- 4.427
- 4.427
- 4.427
- 4.427
- 4.427
- 4.427
- 4.427
- 4.427
- 4.427
- 4.427
- 4.427
- 1.147
- 1.147
- 1.147
- 1.147
- I.147
- 1.147
- 1.147
- 1.147
- 1.147
- 1.147
- 1.147
- 1.147
- 5.919
- 5.919
- 5.919
- 5.919
- 5.919
- 5.919
- 5.919
- 5.919
- 5.919
- 5.919
- 5.919
- 5.919
- 33.236
- 33.236
- 33.236
- 33.236
- 33.236
- 33.236
- 33.236
- 33.236
- 33.236
- 33.236
- 33.236
- 33.236
- 10.005
- 10.005
- 10.005
- 10.005
- 10.005
- 10.005
- 10.005
- 10.005
- 10.005
- 0.818
- 10.361
- 21.092
- 22.926
- 0.762
- 3.408
- 13.432
- 2.923
- 1.772
- 2.801
- 2.041
- 34.495
- 0.818
- 10.361
- 21.092
- 22.926
- 0.762
- 3.408
- 13.432
- 2.923
- 1,772
- 2.801
- 2.041
- 34.695
- 0.818
- 10.361
- 21.092
- 22.926
- 0.762
- 3.408
- 13.432
- 2.923
- 1.772
- 2.801
- 2.041
- 34.695
- 0.818
- 10.361
- 21.092
- 22.926
- 0.762
- 3.408
- 13.432
- 2.923
- 1.772
- 2.801
- 2.041
- 34.695
- 0.818
- 10.361
- 21.092
- 22.926
- 0.762
- 3.408
- 13.432
- 2.923
- 1.772
- 2.801
- 2.041
- 34.695
- 0.818
- 10.361
- 21.092
- 22.926
- 0.762
- 3.408
- 13.432
- 2.923
- 1.772
- 27.669
- 29.688
- 55.479
- 60.814
- 17.853
- 25.708
-31.132
- 28.219
- 26.332
- 18.263
- 16.068
- 64.062
- 2.592
- 14.048
- 23.172
- 32.058
- 2.817
- 6.136
- 16.699
- 5.322
- 4.213
- 5.562
- 5.897
-38.116
- 1.171
- 9.745
- 18.318
- 19.130
- 1.030
- 2.473
- 12.643
- 2.249
- 1.605
- 1.664
- 1.873
- 32.770
- 3.891
- 13.337
- 25.508
- 36.478
- 4.030
-8.166
- 18.818
- 7.232
- 5.439
- 7.552
- 7.116
- 39.411
- 32.630
- 44.360
- 54.626
- 63.925
- 33.203
- 36.045
- 47.223
- 36.369
- 33.097
- 36.506
- 35.804
- 68.563
- 8.557
- 17.452
- 27.081
- 34.627
- 8.887
- 10.380
- 19.890
- 10.120
- 10.068
- 3.969
3.555
- 11.505
- 15.006
5.791
0.582
5.182
-2.414
- 1.678
7.420
8.855
- 6.485
0.451
- 1.162
0.445
- 6.607
0.470
- 0.203
- 0.742
0.126
0.084
- 0.236
- 1.331
- 0.896
- 1.108
- 0.139
2.019
3.041
- 1.023
0.180
0.034
0.081
- 0.588
0.382
- 0.587
1.170
0.944
1.041
- 0.399
- 9.535
0.749
- 0.741
- 1.369
- 0.292
0.350
- 0.734
- 1.058
- 0.699
- 0.478
- 2.665
- 2.200
- 9.665
- 1.107
- 1.303
- 2.457
-2.112
0.009
- 2.371
- 2.429
- 2.534
0.364
1.012
2.114
- 3.598
- 0.022
1.131
1.645
0.906
- 0.193
Table 2 (confi~zued)
Hydrogen-bonding energies
Energy (kcal/mol)
Donor Acceptor Donor Acceptor Complex Bond
NZDA N2A - 10.005 - 2.801 - 11.298 - 0.394
N2DA N3DA - 10.005 - 2.041 - 10.047 0.097
N2DA 03DA - 10.005 - 34.695 - 41.876 0.922
Dielectric constant = 1 .O; water dimer energy = - 1.902.
obtained for the internal energy of the ligand confor-
mation. (6) A weighted sum was performed on the energy
terms to give a final fitness score.
Each gray-coded byte in the binary strings was decoded
to give an integer value between 0 and 255. This integer
value was linearly re-scaled to give a real number between
0 and 27r, which was then used as an angle of rotation, in
radians, for the appropriate rotatable bond. The random-
ised three-dimensional co-ordinates were used as a
starting configuration and the rotations were successively
applied around the rotatable bonds to generate a new set
of co-ordinates for the ligand and protein active site. The
resulting conformations were then passed on to the
least-squares fitting procedure.
For every donor-hydrogen atom and lone pair a virtual
point was created colinear with the bond at 1.4 A from the
donor or acceptor. The integer strings in each chromosome
define mappings that suggest hydrogen bonds between the
protein and ligand. Associated with each hydrogen and
lone pair is a virtual point. Let N be the number of
hydrogen atoms and lone pairs in the ligand, so that
decoding a chromosome gave rise to N pairs containing a
ligand virtual point and a corresponding protein virtual
point. A Procrustes Rotation (Digby & Kempton, 1987)
with a correction to remove inversion, yielded a geometric
transformation that, when applied to all the ligand virtual
points in the N pairs, minimised the least-square distance
between each ligand virtual point and the corresponding
protein virtual point. As not all hydrogen-bonding sites in
the ligand form bonds to the protein, a second
least-squares fit was applied to minimise the djstance
between those pairs of points that were less than 5 A apart.
In order to determine the hydrogen-bonding energy of
the complex each possible combination of donor hydrogen
atom, and lone pair was examined in turn to see whether
or not a bond had actually been formed in the binding
mode resulting from the transformation described above.
The geometrical arrangement of donor, acceptor and both
virtual points was examined, and a weight (between 0 and
1) assigned to the potential bond. The weight was used to
scale the full bond energy between the donor and acceptor
(this full bond energy Epir, is described later), these bond
energies being determined by the modelling experiments.
This weighting scheme was designed to favour hydrogen
bonds that are linear and whose virtual points overlap.
Poor bond angles or long interaction distances gave rise to
weights of zero. The scheme allows configurations that are
not real hydrogen bonds to contribute to the fitness of a
chromosome in the hope that they will evolve to become
full hydrogen bonds (with higher weights and conse-
quently higher fitness values) during the full course of the
run. The geometry of a potential bond is shown in Figure 8.
The weight given to the bond was a function of ci, the
separation of the virtual points, and 0, the bond angle
between the donor hydrogen atoms and the acceptor lone
pairs. Let rot be the weight of the hydrogen bond, such that
zut = distmce-zut + nngkzot. If d was less than 0.2 A then
50
Molecular Recognition of Receptor Sites
Hhdroaen Virtual Point
a -
d ,-Donor
,
t_
P
Lone Pair Virtual Point
6
Acceptor
Figure 8. The geometrical arrangement of the virtual
points representing a lone pair and a hydrogen when a
hydrogen bond is formed. ti is the distance between the
virtual points and 0 is the bond angle between the donor
hydrogen and the acceptor lone pair.
&sfn77ce_zut was 1 aid if d was greater than 3.5 d; then
disnznnce-7ut was 0. Otherwise, d lay in the interval (0.2,3.5),
in which case d was linearly re-scaled to the interval (1,O)
and squared to give distance-wt. Theoretical studies and
observation of crystal structures (Murray-Rust & Glusker,
1984) show that strong hydrogen bonds are obtained if a
line drawn from the donor through the hydrogen atom and
a second line drawn from the acceptor through the lone
pair intersect at around 180, i.e. 0 in Figure 8 should be
close to 180. Accordingly if 0 was greater than 160 then
nrzgle-zut was set to 1 and if 0 was less than 10 then
nngle-zut was set to 0. Otherwise,@ lay in the interval
(160, lo), in which case it was linearly re-scaled to (1,O) and
squared to give angle-rot.
The energy H-Borrd-Energy was a sum of individual
bond energies. Each lone pair in the l&and was compared
with every donor hydrogen atom in the protein active site
and the weights of any bonds formed recorded. For each
&and lone pair, the bond with the highest weight
contributed to H-Band-Energy as did any other bond with
weight greater than 0.4. An identical procedure was
adopted in comparing each donor hydrogen atom in the
ligand with every lone pair in the protein. This mechanism
was developed to allow the GA to form many-centre
hydrogen-bonding motifs, white preventing a large
number of poor bonds making a significant contribution to
the binding energy
Following the placement of the ligand into the active site
of the protein a Lennard-Jones 6-12 potential (Hirschfelder
ef nl., 1964) was used to determine the energy of interaction
between the ligand and protein. The 6-12 potential was of
the form:
where i and j are a pair of atoms, aii is the distance between
i and j divided by the sum of their van der Waals radii, and
kii is the arithmetic approximation to the geometric mean
of ki and ki, where the k values correspond to the minimum
of the potential well between the atom types i and j. The
values for the radii and k values for each atom type were
taken from the SYBYL general purpose Tripos 5.2 force
field (Clark et al., 1989). In order to calculate the interaction
efficiently a lookup table and a cut-off distance, of aii = 1.5,
were employed. A correction was made to the 6-12
potentia1 for atoms involved in hydrogen bonding. The
energy of interaction between a hydrogen atom and
acceptor was set to zero. For the interaction between a
donor and acceptor atom the sum of the van der Waals radii
was scaled by 0.7. The sum of energies for all pairwise
interactions with the addition of any penalty term was then
Complex-VD W-Energy.
The internal steric energy of the ligand conformation
was calculated, using the 6-12 potential described above.
The energy for the ligand, Interflu/-VDW-Energy, was
expressed as a difference in energy between the van der
Waals energy of the current ligand conformation and the
energy of the original minimised conformation (gas-phase)
of the input hgand structure.
The final fitness score was determined by a weighted
sum of all the energy components. The weights were
determined by empirical adjustment to best reproduce
known bound ligand structures. Optimisation of the
weighting scheme is an area of current investigation. Since
higher energies implied poorer solutions, the weights were
all negative. The fitness score was given by:
- H-Band-Energy - 0.0005 x Carrzples-VD W-Energy.
If Ihmal-VD W-Energ!/ was positive then
0.5 x Irz~erlzal-VDW-Elze~gy was subtracted from the
fitness score. In order to prevent the GA from trying to
minimise the internal energy of the ligand below that of the
reference conformation, this term was used only when
Ilzte~rzal-VDW-Elzergy was positive.
The genetic operators
The GA made use of two genetic operators: mutation
and crossover. The crossover operator required two
parents and produced two children, while the mutation
operator required one parent and produced one child.
Operators were chosen using roulette-wheel selection
based on operator weights (Davis, 1991). The operator
weights for crossover and mutation were 10 and 40,
respectively Thus, the GA search procedure emphasised
expIoration rather than exploitation. The parents for these
operators were selected using the technique of roulette-
wheel selection on linear normalised (rank-based) fitness
values (Davis, 1991). The current population was sorted on
increasing fitness scores. The normahsed fitness of the
individual with the worst fitness score in the current
population was set to 25,000. Normalised fitness values
then increased by 12 per individual. This parameterization
equates to a low selection pressure of 1.1, which represents
the relative probability that the best individual will be
chosen as a parent compared with the average individual.
This low selection pressure reduced the likelihood of the
GA converging to sub-optimal solutions.
The crossover operator performed two-point crossover
on the integer strings and one-point crossover on the
binary strings. The one-point crossover on binary strings
is the traditional GA recombination operator. The parent
chromosomes were copied to the children. One of the four
chromosome strings was selected randomly and the
appropriate crossover applied to it. The remaining strings
were unchanged.
The mutation operator performed binary-string mu-
tation on binary strings and integer-string mutation on
integer strings. Each bit in the binary string had a
probability of mutation equal 1.0 over the length of the
binary string. If the binary string remained unchanged
after one application of the mutation operator, the operator
was repeatedly applied until the string was mutated. The
integer-string mutation performed a single mutation on an
integer value. A position was randomly chosen on the
integer string and the value at that position was mutated
Molecular Recognition of Receptor Sites 51
N4 NPW MDA
03DA Pk?DA ER
cY
C-F
C-C- N.l
Cd
C-02
F Nl 02
0.0X2
C-C
td
03A WA oco2
Figure 9. Donor and acceptor types and associated
fragments used in hydrogen-bonding energy determi-
nation.
to a (different) new value that was randomly chosen
from the set of allowed integer values. The operator
proceeded as follows: the parent chromosome was
copied to the child chromosome and one of the four strings
was randomly selected and the appropriate mutation
applied.
The GA automatically terminated following the
application of 80,000 genetic operators. However, conver-
gence (indicated by an improvement of less than 0.01 in the
best fitness score over the previous 6500 operations)
resulted in premature termination.
Calculation of hydrogen-bond energies
The hydrogen-bond energy between a donor and an
acceptor is an important component of the fitness function
since each hydrogen-bonding pair contributes to the
overall energy of binding. Initially the donor (d) and the
acceptor (a) are in solution but on coming together (da)
water (w) is stripped off. Therefore to simulate the
interaction energy Epair is composed of four terms:
S& hydrogen-bond donor and 12 hydrogen-bond
acceptor types were defined based on SYBYL molecular
mechanics atom types (Clark et nl., 1989), These were
embedded in the model fragments shown in Figure 9.
total of 91
C&=o . .
simulation systems (e.g.
. HN + (CH,), or 02A . . N-4) were required to
model the possible pairwise interactions, and the
interaction energy calculated using a number of different
approaches. These included gas-phase semi-empirical
quantum-mechanics (MOPAC, AMI, PM3: Stewart, 1992),
solvent based (AMSOL, SM3: Cramer et al., 1993), VAMP
(Timothy Clark, personal communication) with a self-con-
sistent reaction field (Rinaldi, 1993), and molecular
mechanics (Clark et al., 1989) with MOPAC (AM1 and PM3)
Coulson and Mull&en atom charges (Stewart, 1992). Ab
iuitio calculations are in progress to determine 6-31G*
potential derived charges.
Currently, gas-phase molecular mechanics with PM3
Table 3
Hydrogen-bonding energies for solvated N-4 (lysine)
donor
Energy (kcal/mol)
Acceptor
BR
Donor
1.695
Acceptor
- 0.563
Complex Bond
1.357 - 0.164
NZDA
02
oco2
CL
Nl
N3A
03A
F
N2A
N3DA
1.695 1.328
1.695 0.568
1.695 - 0.260
1.695 - 0.435
1.695 - 0.602
1.695 0.031
1.695 0.068
1.695 - 0.692
1.695 2.533
1.695 - 0.061
3.156 - 0.256
2.571 - 0.081
1.623 - 0.201
1.648 - 0.001
1.709 0.227
1.170 - 0.945
1.535 - 0.617
1.454 0.062
4.167 - 0.450
1.463 - 0.560
03DA 1.695 - 0.850 0.444 - 0.790
Dielectric constant = 78.54; water dimer energy = - 0.389.
Mulliken charges and a dielectric constant of 1.0 in the
receptor cavity give results that are consistent with the
observed binding modes; this may be surprising, however,
recent NMR studies imply that the protein-ligand
hydrogen-bond interactions in the receptor cavity may
have energetics more consistent with gas-phase values
(Beeson et al., 1993) than has previously been appreciated.
Indeed, the local dielectric.may be less significant with
high angular dependence of hydrogen-bonding energies.
The values are listed in Table 2.
The crystal structures studied contained a number of
highly solvated and exposed lysine residues at the lip of
the receptor cavity In order to account for their solvated
nature and, more importantly the highly flexible nature of
the lysine side-chain (resulting in an entropically favoured
solvated state), bonds formed between the ligand and the
charged nitrogen donor in lysine used molecular
mechanics results with a dielectric constant of 78.54 (the
dielectric constant of water at 25C). These energies are
A iI
H- N.plc 6 N.plc
A A
(2)
H. ,C
N.plc
Q
H. ,C. J-l
N.plc N.plc
k A
(3)
Figure 10. The N.plc atom type The protonation on the
nitrogen atom in (1) is shared with the second nitrogen
atom. (2) The N.plc atoms for the fragment in (1). (3) The
N.plc atoms in the arginine residue.
52
Molecular Recognition of Receptor Sites
shown in Table 3. All lysine groups are currently treated as
being solvated, which is rather arbitrary It is intended to
refine this description based on their solvent-exposed
surface areas to determine more accurately which lysine
residues are mobile and therefore which should be
specially treated in this way
A new atom type N.plc, and associated donor type
NPLC, was created to simulate the distribution of charge
across two or three nitrogen atoms, as occurs in
protonated-guanidine groups and arginine residues, such
as illustrated in Figure 10.
The energy of a hydrogen bond A.. . NPLC was
detemlined as follows. Let EN4 be the energy of the bond
A . N4, let ENIJL1 be the energy of the bond A . NPW and
let II be the number of nitrogen atoms across which the
charge is distributed. The bond energy is then:
f (Ep,d + (II - l)Ep&.
Acknowledgements
We thank J. N Champness, C. R. Beddell and A. J Geddes
for useful discussions, the Science and Engineering
Research Council and the Wellcome Foundation Limited
for financial support and Tripos Associates for the
provision of hardware and software. This paper is a
contribution from the Krebs Institute for Biomolecular
Research, which is a designated centre for biomolecular
sciences of the Science and Engineering Research Council.
References
Beeson, C., Nguyen, I., Shipps, G. & Dix, T. A. (1993). A
comprehensive description of the free energy of an
intramolecular hydrogen-bond as a function of
solvation: NMR study J. Amr. Chem. Sot. 115,
6803-6812.
Bernstein, F. C., Koetzle, T. F., Williams, G. J. B., Meyer, F.,
Bryce, M. D., Rogers, J. R., Kennard, O., Shikanouchi,
T. & Tasumi, M. (1977). The Protein Data Bank: a
computer-based archival file for macromolecular
structures. J. Mol. Bid. 112, 535-542.
Blaney J. M. & Dixon, J. S. (1993). A good ligand is hard
to find: automated docking methods. Persyect. Drug
Disc. DCS@I, 1, 301-319.
Blommers, M. J., Lucasius, C. 8.. Kateman, G. & Kaptein,
R. (1992). Conformational analysis of a dinucleotide
photodimer with the aid of a genetic algorithm.
Bio,vohyrwrs, 32, 45-52.
Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C.
& Kraut, J. (1982). Crystal structures of Escherichin co/i
and hctohcilhs cnsei dihydrofolate reductase refined
at 1.7 h; resolution. 1. Biol. Charr. 257, 13650-13662.
Brown, R. D., Jones, G., Willett, l? & Glen, R. C. (1994).
Matching two-dimensional chemical graphs using
genetic algorithms. J. Clw~n. hfom. Cornput. Sci. 34,
63-70.
Champness, J. N., Stammers, D. K., Beddell, C. R. (1986).
Crystallographic investigation of the cooperative
interaction between trimethoprim, reduced cofactor
and dihydrofolate reductase. FEBS Letters, 199,61-67.
Champness, J. N., Achari, A., Ballantine, S. l?, Bryant, I? K.,
Delves, C. J. & Stammers, D. K. (1994). The structure
of ~JlCCWJocytiS Cnriuii dihydrofolate reductase to
1.9 A resolutions. StJwftm, 2, 915-924.
Cheung, H. T. A., Birdsall, B., Frenkiel, T. A., Chau, D. D.
& Feeney, J. (1993). r3C NMR determination of the
tautomeric and ionization states of folate in its
complexes with Lnctobncilllrs cnsei dihydrofolate
reductase. Biocherr~istry, 32, 6846-6854.
Clark, D. E., Jones, G., Willett, P., Kenny, l? W. &Glen, R. C.
(1994). Pliarniacoplioric pattern matching in files of
three-dimensional chemical structures: comparison of
conformational-searching algorithms for flexible
searching. /. C/JUJI. IJI~OIVJI. COJJJ~U~. Sci. 34, 197-206.
Clark, M., Cramer, R. D. & Van Opdenbosch, N. (1989).
Validation of the general-purpose TRIPOS 5.2 force
field. 1. Cornprrt. Clretr~. 10, 982-1012.
Cramer, C. J., Lynch, G. C., Hawkins, G. D. & Truhlar, 0. G.
(1993). AMSOL 3.5~ User Mnr~rml, Quantum Chemical
Program Exchange, Department of Chemistry Univer-
sity of Indiana, Bloomington, IN.
Dandekar, T. & Argos, I? (1993). Folding the main chain of
small proteins with the genetic algorithm. /. Mol. Biol.
236, 844-861.
Davis, J. F., Delcamp,T. J., Prendergast, N. J., Ashford, V. A.,
Freisheim, J. H. & Kraut, J. (1990). Crystal structures
of recombinant human dihydrofolate reductase
complexed with folate and 5-deazafolate. Biochwistry,
29, 9467-9479.
Davis L. D. (1991). Editor of Hmdbook of Gerretic Al~orithrrrs,
Van Nostrand Reinhold, New York.
DesJarlais, R. L., Sheridan, R., Dixon, J. S., Kuntz, I. D. &
Venkataraghavan, R. (1986). Docking flexible ligands
to macromolecular receptors by molecular shape.
1. Med. C~CJJI. 29, 2149-2153.
DesJarlais, R. L., Sheridan, R. P., Seibel, G. L., Dixon J. S.,
Kuntz, I. D. & Venkataraghavan, R. (1988). Using
shape complementarity as an initial screen in design-
ing ligands for a receptor binding site of known three-
dimensional structure. 1. Med. C/ICJ~I. 31, 722-729.
Digby, l? G. N. & Kempton, R. A. (1987). M~rltizwinte
Amdysis Of Ecologicnl COJJllJJlfJ7itiCS. pp. 112-115,
Chapman and Hall, London.
Dixon, J. S. (1993). Flexible docking of ligands to receptor
sites using genetic algorithms. In Trends in QSAR nrxt
Molec~rlnr Mocfelliryq 92 (Wermuth, C. G., ed.),
pp. 412-413, ESCOM, Leiden.
Fountain, E. (1992). Application of genetic algorithms in
the field of constitutional similarity j. ClJe~ll. IJIJOJVJI.
COJII~II~. Sci. 32, 748-752.
Goldberg, D. E. (1989). Genetic A!goriths ill Sen~h,
Oytiraisntiort rind Mnchille Lenmirzg, Addison-Wesley,
Reading, MA.
Goodsell, D. S. & Olson, A. J. (1990). Automated docking
of substrates to proteins by simulated annealing.
ProteilJs: Shwct. FI~JTC~. Genet. 8, 195-202.
Groom, C. R., Thillett, J., North, A. C. T., Pictet, R. &
Geddes, A. J. (1991). Trimethoprim binds in a bacterial
mode to the wild-type and E30D mutant of mouse
dihydofolate reductase. 1. Biol. Cher~r. 266, 19890-
19893.
Hirschfelder, J. O., Curtiss, C. F. & Bird, R. B. (1964).
Molec~dnr T/IcoI?/ ojGnses nJ?d Liquids, John Wiley, New
York.
HO, C. M. W. & Marshall, G. R. (1990). Cavity search: an
algorithm for the isolation and display of cavity like
binding regions. I. CottJput. Aid. Mol. Des. 4, 337-354.
Holland, J. H. (1975). Ahptntiorl irr N~t~lrnl md Artificid
Stjstems, University of Michigan Press, Ann Arbor.
Judson, R. S., Jaeger, E. I?, Treasurywala, A. M. & Peterson,
M. L. (1993). Conformational searching methods for
small molecules: II. Genetic algorithm approach.
1. COJJl/.JUt. CkJJJ. 14, 1407-1414.
Kuntz, I. D. (1992). Structure-based strategies for drug
design and discovery Science, 257, 1078-1082.
Molecular Recognition of Receptor Sites 53
Kuntz, I. D., Blaney, J. M., Oatley, S. J., Langridge, R. &
Ferrin, T. E. (1982). A geometric approach to
macromolecule-&and interactions. J. Mol. Biol. 161,
269-288.
Lawrence, M. C. & Davis, I? C. (1992). CLIX-a search
algorithm for finding novel ligands capable of binding
proteins of known 3-dimensional structure. Proteins:
Strwzt. Rrnct. Gem+. 12, 3141.
Leach, A. R. (1994). Ligand docking to proteins with
discrete side-chain flexibility 1. Mol. Biol. 235,345-356.
Matthews, D. A., Bolin, J. T., Burridge, J. M., Filman, D. J.,
Volz, K. W., Kaufman, B. T., Beddell,C. R., Champness,
J. N., Stammers, D. K. & Kraut, J. (1985). Refined
crystal structures of Escherichin co/i and chicken liver
dihydrofolate reductase containing bound trimetho-
prim. 1. Biol. Chm. 260, 381-391.
Murray-Rust, I? & Glusker, J. (1984). Directional hydrogen
bonding to sp2 and sp3-hybridised oxygen atoms and
its relevance to ligand-macromolecular interactions.
1. Amer. Chern. Sot. 1018-1025.
Payne, A. W. R. &Glen, R. C. (1993). Molecular recognition
using a binary genetic search algorithm. 1. Mol. Graph.
11, 74-91.
Rinaldi, D., Rivail, J. & Rguini, N. (1993). Fast geometry
optimisation in self-consistent reaction field compu-
tations on solvated molecules. 1. Conyut. Chetn. 13,
675-680.
Shoichet, B. K., Stroud, R. M., Santi, D. V, Kuntz, I. D. &
Perry K. M. (1993). Structure-based discovery of
inhibitors of thymidylate synthase. Science, 259,
1445-1450.
Smellie, A. S., Crippen, G. M. & Richards, W. G. (1991).
Fast drug-receptor mapping by site-directed
distances: a novel method of predicting new
pharmacological leads. J. Chem. Inform. Comprtt. Sci.
31,386-392.
Stammers, D. K., Champness, J. N., Beddell, C. R., Dann,.
J. G., Eliopoulos, E., Geddes, A. J., Ogg, D. & North,
A. C. T. (1987). The structure of mouse L1210
dihydrofolate reductase-drug complexes and the
construction of a model of human enzyme. FEBS
Letters, 218, 178-184.
Stewart, J. J. I? (1992). MOPAC User Mnnnnl Version
6.0, Quantum Chemical Program Exchange, Depart-
ment of Chemistry, University of Indiana, Blooming-
ton, IN.
Vermersch, I? S., Lemon, D. D., Tesmer, J. J. G. & Quiocho,
F. A. (1991). Sugar-binding and crystallographic
studies of an arabinose-binding protein mutant
(metLeu) that exhibits enhanced affinity and altered
specificity Biochnnistiy 30, 6861-6866.
von Itzstein, M., Wu, Wen-Yang, Kok G. B., Pegg, M. S.,
Dyson, J. C., Jin, B., Phan, T. V, Smythe, M. L., White,
H. F., Oliver, S. W., Colman, I? M., Varghese, J. N.,
Ryan, D. M., Woods, J. M., Bethell, R. C., Hotham, V. J.,
Cameron, J. M. & Penn, C. R. (1993). Rational design
of potent sialidase-based inhibitors of influenza virus
replication. N&we (London), 363, 418-423.
Edited by F. Cohen
(Received 10 May 1994; accepted 30 August 1994)