1). The document describes an in silico structure-activity relationship study that models the evolution of drug resistance in Plasmodium falciparum dihydrofolate reductase (DHFR) by introducing amino acid substitutions at positions 45 and 55. 2). Different amino acid substitutions were introduced in silico and their effects on resistance to various antifolate drugs were predicted using molecular modeling and docking simulations. 3). Substitutions at position 45 generally conferred resistance to select drugs like WR99210 and P218 while substitutions at position 55 often resulted in broader resistance, with M55F and M55H showing pan-resistance.

1. Predictive Structure-Based Models of Evolved Drug Resistance
Alissa Calderon a,b*, Carla Islas b, Robert P. Metzger a, Gary B. Fogel c, David Hecht a,b
a. Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182
b. Department of Chemistry, Southwestern College, Chula Vista CA 91910
c. Natural Selection, Inc., San Diego, CA 92121
Background
References
Methods
This research was supported by the National Institute of General Medical Sciences of the National Institutes
of Health under Award Number SC3GM100791. The content is solely the responsibility of the authors and
does not necessarily represent the official views of the National Institutes of Health.
The evolution of drug resistance in malaria
continues to be a widespread concern. Many anti-
malarial drugs target key proteins such as
dihydrofolate reductase (DHFR). However in
malaria, the structural plasticity of DHFR allows it to
maintain its active site and catalytic activity, while
resisting drug binding. One way to better
understand this process is through the use of in
silico evolution as a way to predict and model
likely amino acid changes in the active site.
In previous studies, several rounds of in silico
evolution were performed with Plasmodium
falciparum (Pf) DHFR. Through these studies, the
amino acid substitutions V45T and M55I were
identified for potentially conferring resistance to one
or more of the following anti-folate drugs:
pyrimethamine, cycloguanil, methotrexate,
trimethoprim, P65, P218, and WR99210.
Presented here is an in silico structure-activity
relationship (SAR) study in which different amino
acid substitutions were made at DHFR positions 45
and 55. The purpose of this study is develop a
better understanding of the structural basis behind
a pathogen’s development of drug resistance.
Results
1). Hecht et al. (2012) “Modeling the evolution of drug resistance in
malaria”, JCAMD, 26:1343-1353.
2). Hecht et al. (2012) “Towards predictive structure-based models of
evolved drug resistance”, 2012 IEEE Symposium on
Computational Intelligence in Bioinformatics and Computational
Biology, San Diego, pp 120–126.
3). Fogel et al. (2013) “Modeling the Evolution of Drug Resistance in
Plasmodium falciparum”, 2013 IEEE Congress on Evolutionary
Computation, Cancun, Mexico.
 Sequence Variation
Using the BioEdit software package, amino acid
substitutions were introduced into the wt-Pf-DHFR
sequence at positions 45 and 55. (See Table 1).
These sequences were saved in FASTA format and
imported into MOE (www.chemcomp.com).
 Homology Modeling
Homology models were generated using MOE with
default settings using 1J3I.pdb as a structural
template (wt-PF-DHFR x-ray crystal structure). All
structures had rmsd values <1Å.
 Fitness & Selection
Docking experiments were performed using GOLD
(www.ccdc.cam.ac.uk). Each sequence was scored
using a fitness function specifically designed
specifically to evaluate the ability of each sequence
to maintain binding affinity for the co-factor NADPH
and the substrate dihydrofolate (DHF) while reducing
affinity for the inhibitor:
i). Docked conformations of NADPH and 7,8 dihydrofolate
match x-ray conformations.
ii). Docked conformations of the inhibitor do not bind in
the active site pocket. Poses found to dock outside of
the pocket by visual inspection satisfy this constraint.
iii). Docking scores of NADPH and 7,8-dihydrofolate for
each offspring sequence should be roughly similar to
that of the parent sequence (as well as to that of the wild
type DHFR sequence). Lower docking scores imply loss
of binding affinity resulting in a selective disadvantage.
Methods
Position 45:
Interestingly, residue V45 is not in the substrate
binding pocket. It can be hypothesized that amino
acid substitutions at this position will cause
conformation changes in the adjacent amino acids
(upon folding), in particular L46, that will result in
resistance.
To test this hypothesis a series of 6 different amino
acids: G, A, C, I, M and N were tested. Interesting
patterns of specific resistance were observed.
• In particular – all amino acid substitutions were
predicted to be non-resistant to trimethoprim and
methotrexate which are very similar in structure to
the substrate DHF.
• All amino acid substitutions resulted in resistance
to WR99210 and most to the experimental
compound P218.
Also interestingly V45T, V45G and V45C were
predicted to be resistant to pyrimethamine but not to
cycloguanil.
Figure 1. Workflow for generating and evaluating variant
DHFR sequences. The initial input is the wt Pf-DHFR
sequence. The loop of variation, scoring, and, generation
of parent solutions for the next “generation” of evolution
continues until a termination criterion is satisfied [1-3].
Figure 2. Superposition of x-ray crystal conformations of
trimethoprim and NADPH bound to wt Pf-DHFR from
3FRB.pdb (colored in gold) vs. docked conformations (in
CPK), RMSD values <1.00Å.
Table 1. SAR data for amino acid substitutions were made at positions 45 and 55 are presented below.
Mutation Pyrimethamine Cycloguanil Trimethoprim Methotrexate WR99210 P65 P218
Wt NON NON NON NON NON NON NON
V45A NON NON NON NON resistant NON NON
V45C resistant NON NON NON resistant NON resistant
V45G resistant NON NON NON resistant NON NON
V45I NON NON NON NON resistant NON resistant
V45M resistant resistant NON NON resistant NON resistant
V45N NON NON NON NON resistant NON resistant
V45T resistant NON NON NON resistant NON resistant
M55I resistant resistant NON NON resistant NON resistant
M55F resistant resistant resistant resistant resistant resistant resistant
M55H NON resistant resistant resistant resistant resistant resistant
M55Q NON NON NON resistant NON NON resistant
Discussion
Position 55:
Unlike V45, residue M55 is in the substrate
binding pocket.
Not surprisingly, M55F and M55H resulted in
predicted pan-resistance. Both amino acids have
large, bulky side chains that prevent the inhibitors
from binding.
M55I was predicted to be resistant to both
pyrimethamine and cycloguanil, but not to
trimethoprim as well as methotrexate.
M55Q was predicted to be resistant only to
methotrexate and P218.
Future Work:
Additional in silico as well as experimental
validation studies are planned.