Discovery of new antimalarial chemotypes through chemical methodology and library development

Article abstract of DOI:10.1073/pnas.1017666108

In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC₅₀'s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.

Full text of DOI:10.1073/pnas.1017666108

Discovery of new antimalarial chemotypes through
chemical methodology and library development
Lauren E. Browna,1, Ken Chih-Chien Chengb,1, Wan-Guo Weia,1, Pingwei Yuana,1, Peng Daia, Richard Trillesa, Feng Nia,
Jing Yuanc, Ryan MacArthurb, Rajarshi Guhab, Ronald L. Johnsonb, Xin-zhuan Suc, Melissa M. Domingueza,
John K. Snydera,2, Aaron B. Beelera,2, Scott E. Schausa,2, James Ingleseb,2, and John A. Porco, Jr.a,2
aDepartment of Chemistry and Center for Chemical Methodology and Library Development (CMLD-BU), Boston University, 590 Commonwealth Avenue,
Boston, MA 02215; bNIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892; and
cLaboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892
Edited by Stuart L. Schreiber, Broad Institute, Cambridge, MA, and approved February 25, 2011 (received for review November 29, 2010)
In an effort to expand the stereochemical and structural complexity
of chemical libraries used in drug discovery, the Center for Chemical
Methodology and Library Development at Boston University has
established an infrastructure to translate methodologies accessing
diverse chemotypes into arrayed libraries for biological evaluation.
In a collaborative effort, the NIH Chemical Genomics Center deter-
mined IC50’s for Plasmodium falciparum viability for each of 2,070
members of the CMLD-BU compound collection using quantitative
high-throughput screening across five parasite lines of distinct
geographic origin. Three compound classes displaying either differ-
ential or comprehensive antimalarial activity across the lines were
identified, and the nascent structure activity relationships (SAR)
from this experiment used to initiate optimization of these chemo-
types for further development.
supporting identification of low to moderately active chemotypes
to maximally leverage the library. Herein, we report the initial re-
sults employing this technique to characterize the antimalarial
activity of a CMLD-BU compound library and the activities iden-
tified from three distinct chemical series.
Results and Discussion
CMLD-BU Compound Collection. In 2009, the CMLD-BU library
consisted of 2,070 unique chemical structures which were derived
from approximately 18 synthesized libraries and 10 diversity
collections. To categorize the CMLD-BU compound collection,
we evaluated the complexity as defined by stereogenenic carbon
content and sp3∕sp2 character (11). In this regard, we also com-
pared our collection to the NIH Molecular Libraries Small-
Molecule Repository (MLSMR). The analysis revealed a signifi-
cant difference in overall complexity as defined by the ratio of
stereogenic carbons to total carbons as well as total stereogenic
carbon count. For instance, the CMLD-BU compounds have an
average ratio of stereogenic carbons of about 0.1, while analysis
of the MLSMR indicates an average of approximately 0.02. Like-
wise, the average number of steoreogenic carbons per molecule in
the CMLD-BU collection is 2.8, while the average number in the
MLSMR is 0.4.
chemical biology ∣ organocatalytic ∣ polycyclic ketal
he synthesis of compound libraries embodying structural
T
diversity and complexity is one potential avenue to enabling
the discovery of potent and selective bioactive agents (1–3). Syn-
thetic methodology aimed at library design and distribution is the
focus of the Center for Chemical Methodology and Library Devel-
opment at Boston University (CMLD-BU). Currently the CMLD-
BU maintains a collection of approximately 2,500 compounds
derived from reaction methodologies developed in the Center
and associated groups. The CMLD-BU has developed a workflow
that leverages focused methodology development (4) and identi-
fication of new scaffolds and chemical reactions utilizing multi-
dimensional reaction screening (5) for the synthesis of libraries.
Another method for structural characterization of the CMLD-
BU compound collection is based on an approach introduced
by Sauer and Schwarz utilizing principle moments of inertia to
describe the overall three-dimensional shape of molecules (12).
More recently, efforts by Clemons and Schreiber have shown this
In a collaboration between the CMLD-BU and the NIH Che- method to be useful for description and comparison of compound
mical Genomics Center (NCGC), an effort to discover unique che-
motypes which inhibit the viability of P. falciparum, the causative
agent of malaria, is underway. Malaria not only inflicts tremendous
suffering and morbidity on the human population, but has drama-
tically influenced the course of civilization with profound sociolo-
gical and economic implications (6). In response, there have been
significant screening efforts for new antimalarial agents (7, 8). Ef-
fective treatments continue to be needed in part due to acquired
resistance of P. falciparum. Identification of molecular targets
affecting the viability of malaria is necessary for efficient drug de-
velopment as is the discovery of chemotypes active across a variety
of geographic isolates. With an aim to improve the process of iden-
tifying new compounds and targets, we have used quantitative
high-throughput screening (qHTS) to achieve pharmacological
fingerprints of chemical libraries (9). This technique tests each
compound as a seven point titration covering four orders of mag-
nitude in concentration thus allowing sufficient resolving power by
virtue of IC50 determinations to reveal subtle differences in com-
pound activities between parasite lines. A compound with a differ-
ential chemical phenotype (DCP) can enable mapping of target
genes using genetic recombination wherein the DCP emerges
between two or more lines which represent the parental parasites
of a genetic cross (10). Additionally, qHTS reveals nascent SAR
collections (13). A similar analysis of the CMLD-BU compound
collection shows that the overall shape is heavily “spherical” in
nature with significant degree of “rod-like” character (Fig. 1).
Overall, the CMLD-BU collection does not have a significant
representation of “flat” compounds, correlating with a high sp3
content within the collection. This shape distribution distin-
guishes the CMLD-BU collection from those typically found in
industry or commercial collections (14).
Author contributions: J.K.S., A.B.B., S.E.S., J.I., and J.A.P.J. designed research; L.E.B.,
K.C.-C.C., F.N., W.-G.W., P.Y., R.T., J.Y., P.D., R.M., R.L.J., X.S., and M.M.D. performed
research; R.M. and R.G. analyzed data; and J.K.S., A.B.B., S.E.S., J.I., and J.A.P.J. wrote
the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The chemical compounds reported in this paper have been deposited in
PubChem (AIDs 504350, Dd2 504314, GB4 504315, 7G8 504316, HB3 504318, CP250
504320).
1L.E.B., K.C.-C.C., W.-G.W., and P.Y. contributed equally to this work.
2To whom correspondence may be addressed. E-mail: porco@bu.edu or jinglese@
mail.nih.gov.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1017666108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1017666108
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control inhibitors, artemisinin and mefloquine, were present on
every plate as full intraplate titrations and exhibited the expected
IC50 values (see SI Appendix, Table S1). The compound library
was also counterscreened for color interference to eliminate po-
sitives that might have resulted from light absorbance interfer-
ence-based artifacts (see SI Appendix, Fig. S1). Data from this
screen was deposited into PubChem (Summary AID ¼ 504350
http://www.ncbi.nlm.nih.gov/pccompound) and are summarized
in SI Appendix, Table S2.
Primary screening results identified three chemotypes (1–3)
which inhibited proliferation either nonselectively (1 and 2) or
differentially (3), having completely fit concentration-response
curves (CRCs) displaying full efficacy (curve class 1a), or partially
complete CRCs (class 2a) (9). To further validate activities, com-
pounds were retested in an intraplate titration format using
twelve-point, threefold dilutions of the compounds to obtain
more accurate IC50 values (Fig. 2; SI Appendix, Table S5) (vide
infra). These compounds were then counterscreened in an ery-
throcyte viability assay to rule out the possibility that positive ac-
tivity resulted from erythrocyte toxicity rather than parasite
death. The results confirmed that all the identified positives
(compounds associated with curve classes 1a, 1b, and 2a) inhib-
ited parasite growth. Little or no erythrocyte toxicity and/or color
interference was observed (see SI Appendix, Table S5, Fig. S1).
Fig. 1. Principle moments of inertia analysis of the CMLD-BU compound
collection.
Quantitative High-Throughput Screening for Inhibitors of P. falcipar-
um Proliferation. Utilizing a SYBR green I dye DNA staining assay
(15) to measure proliferation of P. falciparum in human erythro-
cytes, we conducted qHTS across five geographically distinct
clonal lines of the parasite (CP250, Dd2, HB3, 7G8 and GB4) uti-
lizing the CMLD-BU small-molecule library (2,070 compounds).
We selected these five lines to include two sets of parents of two
genetic crosses (Dd2xHB3, and 7G8xGB4) for which we have pro-
geny available to permit genetic mapping of the chromosomal
locus controlling the differential activity (10). The CP250 line from
Cambodia was included to identify compounds active on a multi-
drug resistant strain (16).
Identification of Nonselective Chemotypes. Among the 2,070 com-
pounds tested, several associated with curve classes 1a-2a (SI
Appendix, Table S2) were found to be pan-active with IC50 values
less than 2 μM across all the five parasite lines. In addition, these
compounds also showed no significant effect on the viability of
HepG2 and HEK293 cells at concentrations effective (IC50) in
inhibiting P. falciparum viability (SI Appendix, Table S5). Structur-
al analysis revealed that these pan-active compounds were de-
rived from polycyclic ketal and indoline alkaloid chemotypes
(1 and 2, Fig. 2).
Initially, each compound was tested in qHTS format at seven
fivefold interplate dilutions beginning at 5.7 μM. The assay as
measured by standard methods displayed acceptable performance
for each parasite line (Z′ factor ≥0.7 and a S∶B ratio ≥6). Two
Polycyclic ketals. We have described the synthesis of polycyclic
ketals which were discovered through the process of multidimen-
Fig. 2. Active series from the CMLD-BU compound collection vs. ability to inhibit the viability of five geographic P. falciparum lines. Scaffolds of active series,
polycyclic ketal (1), indoline alkaloid (2), and dihydropyrimidinone (3), and activity of representative compound as identified in qHTS (A–C) and
follow-up confirmation (D–F). Plots show the compound potency against five geographic P. falciparum lines (square, 7G8; up triangle, Dd2; down
triangle GB4; diamond, HB3; CP250 open circle) as determined in the SYBR Green-based viability assay in qHTS (A–C) and in a 12-point follow-up
titration (D–F).
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Table 1. Active polycyclic ketals
Structure
7G8 GB4
HB3 Dd2
CP250
2.5 μM
3.8 μM
(0.6, n ¼ 2)
(0.6, n ¼ 4)
8.7 μM
(0.7, n ¼ 2)
6.5 μM
4.0 μM
(0, n ¼ 2)
(0.2, n ¼ 4)
1.7 μM
2.9 μM
(0.1, n ¼ 2)
(0.9, n ¼ 2)
na
1.3 μM
(0.1, n ¼ 2)
9.0 μM
(1.5, n ¼ 2)
1.0 μM
2.5 μM
(0.2, n ¼ 2)
(0, n ¼ 2)
6 μM
(0.5, n ¼ 2)
4.2 μM
(0.4, n ¼ 2)
1.9 μM
(0.5, n ¼ 2)
Results are reported as effective concentration values needed to inhibit
parasite growth at 50% (IC50) in μM. Standard deviation and number of
replicates are reported in parentheses. Replicate numbers >2 represent
assays carried out under identical conditions on different days.
Compounds were retested in an intraplate titration format using twelve-
point, threefold dilutions.
Fig. 3. General cycloisomerization/annulation route to polycyclic ketals and
polycyclic ketal library building blocks.
sional reaction screening (5). The polycyclic ketal core 6 is
derived from cycloisomerization/annulation of o-alkynyl benzal-
dehydes (4) and 1,3-dicarbonyls such as 1,3-cyclohexanedione 5
in the presence of a cationic rhodium (I) catalyst (Fig. 3).
Upon discovery of this polycyclization reaction, we explored the
scope and limitations regarding the dicarbonyl component. We
chose a series of 24 1,3-dicarbonyl reaction partners which were
reacted with alkynyl benzaldehyde 4. Each reaction was carried
out under optimized reaction conditions (10 mol% RhðcodÞ BF4,
C6H5Cl, 80 °C, 4 h). Overall, a broad scope of 1,3-dicar2bonyl
substrates participated in the polycyclic ketal formation affording
moderate to good yields rendering the methodology ideal for
library synthesis.
We next designed an array of 192 polycyclic ketals using sixteen
alkynyl benzaldehydes (derived from cross coupling of four ortho-
bromo benzaldehydes and four terminal alkynes) and twelve 1,3-
dicarbonyls as building blocks (Fig. 3). Library synthesis was con-
ducted under optimized conditions (10 mol% RhðcodÞ2BF4,
C6H5Cl, 80 °C, 12 h) on a 4 mmol scale. Products were isolated
by mass-directed, preparative high-performance liquid chromato-
graphy (HPLC). Sixty reactions had an isolated yield of 70% or
greater and fifty reactions had isolated yields of 10–70%. The
remaining reactions did not afford isolatable products. Purities
of the isolated compounds were generally high (>90%) as mea-
sured by HPLC analysis. Library members which were isolated
and maintained high purity (90 compounds) were included in the
collection of 2,070 compounds tested for biological activity.
Activity profiling of the screened polycyclic ketal library mem-
bers revealed a number of active compounds ranging from very
low to moderate activity. Validation of the most active com-
pounds (1a and 1b) afforded good dose-response curves although
with reduced potency in comparison to the primary screen
(Table 1). We synthesized a number of analogs with variation
of the alkyne derived substitution and 1,3-dicarbonyl. Among
these derivatives, we identified compound 1c as the most active.
Compounds 1a and 1c were not cytotoxic to HepG2, HEK293,
and red blood cells (SI Appendix, Table S5). Although these poly-
cyclic ketals have low μM activity against the five lines, they re-
present a unique class of potential antimalarial chemotypes. We
are currently synthesizing analogs in an attempt to explore addi-
tional SAR and increase antimalarial potency.
Indoline alkaloids. Indoline alkaloid scaffolds are derived from
intramolecular inverse-demand Diels-Alder cycloaddition of
tryptophan derivatives tethered to triazines (Fig. 4) (17). The un-
derlying approach is representative of a methodology focused on
the synthesis of scaffolds which resemble Aspidosperma alkaloid
(18) natural products. Libraries based on three scaffolds were
synthesized using a general synthetic approach. A representative
scaffold synthesis begins with Boc-protected tryptophan (7). De-
protection and subsequent nucleophilic aromatic substitution of
triazine 8 afforded the inverse-demand Diels-Alder precursor 9.
In situ acylation [lowering the triazine lowest unoccupied mole-
cular orbital (LUMO)] and thermolysis promoted cycloaddition
with loss of nitrogen to afford the protected indoline alkaloid
scaffold 10 which could be readily deprotected to scaffold 11.
A library of 199 related indoline alkaloids derived from scaffold
12–14 were synthesized via N14 diversification employing acyl
and sulfonyl chlorides (19).
Of the 199 indoline alkaloid scaffolds present in the library, a
number of weakly active compounds clustered to scaffold 12.
Compounds 2a and 2b (Table 2) were found to be the most active
against all five lines. Initial SAR indicates that the activity ap-
pears to be particular to structures having an aryl sulfonamide
Fig. 4. General route to indoline alkaloid scaffolds.
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Table 2. Active indoline alkaloids
cal phenotypes (DCPs) by selectively acting on one of two par-
ental lines (e.g., 7G8 or GB4) can enable the identification of
genes underlying the DCPs through genetic mapping methods
(10). In the CMLD-BU collection, we identified only six potential
DCP’s having >5 fold differential activity between any two lines
(SI Appendix, Table S5) which were all derived from dihydropyr-
imidone scaffolds (15). Dihydropyrimidin-2(1H)-ones exhibit
wide-ranging biological activity. Chiral hetereocyclic compounds
such as monastrol (20, 21) and SNAP-7941 (22) are active as
single enantiomers. We have developed an organocatalytic route
to these compounds using the Cinchona alkaloids, chiral bases
derived from the Cinchona tree (Fig. 5) (23, 24). Because the
Cinchona catalysts are available in pseudoenantiomeric form
from natural sources, both enantiomers of dihydropyrimidinones
are easily accessible. In a demonstration of utility, the method
was used in the construction of a library (24) of chiral dihydro-
pyrimidones 15.
In designing synthetic routes for further elaboration of these
chemical structures, we were inspired by naturally occurring
guanidines (25) such as the batzellidines and derived analogs,
inhibitors of HIV gp120-human CD4 binding (26) and HIV-1
Nef-protein interactions (27) and the muramycins, antimicrobial
natural products active against Gram-positive bacteria (28, 29). A
synthetic route was devised converting the dihydropyrimidinone
15 to a collection of guanidines 17 via the thiourea 16 (Fig. 5).
Conversion of the dihydropyrimidinone to the thioxodihydro-
pyrimidine was accomplished using Lawesson’s reagent (30).
Alkylation of the sulfur using CH3I, followed by substitution with
amines, afforded the desired guanidines in a two-step process
(31). Viability of the route was demonstrated on nine dihydropyr-
imidinones utilized to prepare 96 guanidines. After purification
by mass-directed HPLC, 81 guanidines were tested for biological
activity.
Structure
7G8 GB4
HB3 Dd2
CP250
1.7 μM
1.3 μM
(0.7, n ¼ 8)
(0.5, n ¼ 14)
1.5 μM
(0.5, n ¼ 6)
1.2 μM
1.1 μM
(0.5, n ¼ 8)
(0.5, n ¼ 14)
2.5 μM
1.6 μM
(0.2, n ¼ 2)
(0.3, n ¼ 4)
2.3 μM
(0.0, n ¼ 2)
1.7 μM
(0.1, n ¼ 2)
1.6 μM
(0.09, n ¼ 4)
0.7 μM
0.6 μM
(0.1, n ¼ 4)
(0.09, n ¼ 4)
0.6 μM
(0.0, n ¼ 2)
0.6 μM
0.6 μM
(0.07, n ¼ 4)
(0.03, n ¼ 4)
0.8 μM
0.6 μM
(0.07, n ¼ 6)
(0.0, n ¼ 6)
0.6 μM
(0.05, n ¼ 2)
0.6 μM
0.6 μM
(0.1, n ¼ 6)
(0.1, n ¼ 6)
Results are reported as effective concentration values needed to inhibit
parasite growth at 50% (IC50) in μM. Standard deviation and number of
replicates are reported in parentheses. Replicate numbers >2 represent
assays carried out under identical conditions on different days.
Compounds were retested in an intraplate titration format using twelve-
point, threefold dilutions.
During the course of screening, these compounds were iden-
tified as possessing antimalarial activity. More importantly, a
differential chemical phenotype was observed among the GB4,
7G8, Dd2, and HB3 lines (SI Appendix, Table S5) for which the
progeny of two crosses are available. Fig. 6 depicts a two-dimen-
sional heat map of activity where compounds were first ranked by
IC50 in the GB4 strain followed by clustering of the chemical
structures. Two structurally similar compounds were identified
as possessing significant differential activity; 3a and 3b with a
7G8 IC50∕GB4 IC50 (selectivity factor) of 17 and 10 (See Pub-
Chem AIDs 504316 and 504315), respectively and sufficient to
allow genetic mapping.
substitution at N14 and benzyl substitution at the indole nitrogen.
Validation of compounds 2a and 2b recapitulated the preliminary
observation of dose-response curves with steep Hill coefficients
(Fig. 2). The compounds were not adversely cytotoxic to HepG2,
HEK293, or red blood cells at their antimalarial IC50’s (SI
Appendix, Table S5).
Based on the apparent preliminary SAR obtained, we synthe-
sized analogs directed at improving potency for pan-active indo-
line alkaloid scaffolds. Our first observation was that substitution
of the triazine methyl esters for ethyl esters reduced activity (SI
Appendix, Table S5). However, due to solubility and stability is-
sues, we chose to synthesize a series of forty ethyl ester analogs
varying substitution of both the aryl sulfonamide and indole ni-
trogen. The most active compounds were those bearing a o-tri-
fluoromethyl benzyl group at the indole nitrogen. Interestingly
scaffolds having either p-bromo or p-nitrile-substituted benzyl
groups were less active (SI Appendix, Table S5). The most active
compound in this series was 2c bearing a 3,5-dimethylisoxazole
sulfonamide moiety. Phenyl sulfonamides tolerated para substitu-
tion (2d and 2e) with a decrease of activity observed upon ortho
substitution (SI Appendix, Table S5). These analogs were also not
cytotoxic to HepG2, HEK293, or red blood cells in the range ef-
fective at inhibiting parasite growth (SI Appendix, Table S5).
Utilizing an efficient complexity building inverse-demand
Diels-Alder reaction, we were able to synthesize natural product-
inspired scaffolds which were utilized in library synthesis. The
resulting indoline alkaloid compounds were found to inhibit
growth of a number of P. falciparum lines. Initial follow-up has
shown improvement of potency through analog synthesis. Contin-
ued studies are focused on further SAR and molecular target
identification.
The original guanidine library was a demonstration library
containing only a fraction of the full combinatorial design. Once
Fig. 5. (A) Organocatalytic asymmetric synthesis of dihydropyrimidinone
scaffolds. (B) Synthesis of DHP-derived guanidine library. Reagents: (i) 2
equivalent Lawesson’s reagent, PhCh3, reflux, 48 h. (ii) 20 equivalent CH3I,
CH3CN, 75 °C, 2 h. (iii) R5-NH2, CH3CN, 83 °C, 36 h.
Identification of an Isolate Selective Chemotype. In previous studies,
we demonstrated that compounds displaying differential chemi-
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Fig. 6. Heat map analysis of DHP compounds. (A) Results were ranged in order of effective concentrations necessary to inhibit proliferation at 50% (IC50) in
the GB4 line. The resulting structures were then clustered and fold selectivity calculated for IC50 (B) Magnification of most active compounds.
(C) Substructure analysis of the most active compounds. (D) Compounds 3a and 3b exhibiting the highest level of 7G8 IC50∕GB4 IC50 selectivity.
.
these initial leads were retested, a second synthetic effort was
undertaken to elaborate structure activity relationships. A com-
binatorial library of 630 compounds was designed and is currently
undergoing evaluation in the antimalarial assays. From results
obtained to date, this compound class possesses intriguing biolo-
gical properties against malaria that offer significant potential for
further development (Table 3). First, the enantiomers of 3b were
independently prepared and assessed in the assays for differential
activity. The R-enantiomer 3c exhibited greater activity in the
GB4 line than the S-enantiomer 3d with a selectivity factor of
48 (entries 1 and 2, Table 3). More interestingly, compounds with
significantly greater activity against GB4 and selectivity factors
were identified (compounds 3e–3g, entries 3–5, Table 3). An in-
itial statement about active substituents include electron deficient
aromatic groups with substitution at the meta- or para- positions;
the most active to date being compound 3e with an IC50 of 20 nM
against GB4 and a selectivity factor of 275. While certainly at the
initial stages of development, this compound design should prove
suitable for identifying mechanisms of resistance and possibly
unique drug target identification. Current efforts are directed
toward attaining these objectives.
A series of dihydropyrimidinone guanidines was identified lead-
ing to compounds with low nM IC50’s and high selectivity for
the GB4 line. These initial efforts should pave the way for future
projects directed at further development of unique antimalarial
agents as well as identification of their molecular targets.
Table 3. Guanidine analogs
Entry
1
Structure
7G8
GB4
7G8∕GB4*
9.5 μM
(0.6, n ¼ 4)
0.2 μM
(0.06, n ¼ 4)
48
9.8 μM
2.8 μM
2
3
4
4
(0.6, n ¼ 4)
(0.3, n ¼ 4)
5.5 μM
0.02 μM
Conclusion
275
135
(0.81, n ¼ 8) (0.04, n ¼ 8)
The current work illustrates the developing paradigm for utilizing
synthetic methodologies for the synthesis of libraries of complex
chemotypes rich in stereochemical and structural features. Once
prepared, these libraries can be examined in a host of biological
processes, here exemplified with a neglected disease of the third
world for discovery of probes and future drugs. In our collabora-
tive effort, the CMLD-BU compound collection was assayed for
antimalarial activity against five lines of P. falciparum with distinct
geographic origins, representing genotypic variation in part
resulting from acquired antimalarial drug resistance (16). Over-
all, the CMLD-BU compound collection provided an effective
source of chemotypes in the antimalaria assays. The collection
yielded 1–4% high quality actives defined as those giving qHTS
curve classes 1a, 1b, or 2a (9) suitable for extraction of prelimin-
ary SAR. Specifically, we identified three compound classes
which variously inhibit the growth of the P. falciparum lines tested
in this study. Two compound classes in this library, polycyclic
ketals and indoline alkaloid structures, were found to be active
against multiple geographic parasite lines with moderate activity.
5.4 μM
0.04 μM
(0.08, n ¼ 8) (0.06, n ¼ 8)
2.8 μM
(2.0, n ¼ 8)
0.04 μM
(0.04, n ¼ 8)
5
70
Results are reported as effective concentration values needed to inhibit
parasite growth at 50% (IC50) in μM
*7G8 IC50∕GB4 IC50. Standard deviation and number of replicates are reported
in parentheses. Replicate numbers >2 represent assays carried out under
identical conditions on different days. Compounds were retested in an
intraplate titration format using twelve-point, threefold dilutions.
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library was screened against each parasite line at seven fivefold dilutions
beginning at 5.7 or 29 μM. The antimalarial drugs artemisinin, mefloquine,
and DMSO were included as positive and negative controls for each plate,
respectively. The primary qHTS results are available in PubChem under the
following Assay Identification Descriptions (AIDs); Dd2 504314, GB4 504315,
7G8 504316, HB3 504318, CP250 504320.
Materials and Methods
Cycloisomerization/Annulation. Detailed procedures for the synthesis of
polycyclic ketals, characterization of alkynyl benzaldehydes, description of
representative library members, and biologically active polycyclic ketas are
provided in the SI Appendix.
Indoline Alkaloids. Detailed procedures for the synthesis of indoline alkadoids
are outlined in previous publications (14, 16). Characterization of biologically
active indoline alkaloids are provided in the SI Appendix.
Mammalian Cell Viability Assays. Cell viability assays for HEK293 and HepG2
lines, and human red blood cells were carried out using Cell TiterGlo (Prome-
ga) according to manufacturer’s instructions. Assays were performed in solid
white 1,536-well plates (Nexus Biosystems) and measurements made on a
ViewLux (Perkin Elmer). See protocol SI Appendix, Table S3 for additional
details.
Guanidine Library Preparation. Guanidines were prepared from the
corresponding thioxodihydropyrimidines. Enantioenriched thioxodihydro-
pyrimidines were obtained via treatment of enantioenriched dihydropyrimi-
dinones (24) with Lawesson’s reagent (30). Racemic thioxodihydropyrimi-
dines were obtained according to the procedure of Ryabukhin, et al (32).
Guanidines were synthesized according to the method of Taylor and Cain,
via formation of the thiomethyl ether followed by nucleophilic displacement
of thiomethane (33, 34). See SI Appendix.
Optical Interference Assay. Potential compound interference with the SYBR
green I dye viability assay was determined by adding test compound follow-
ing a 72 h incubation time and reading fluorescence intensity at 485∕535 nm
excitation (EX)/emission (EM) on an EnVision (Perkin Elmer). See protocol SI
Appendix, Table S4 for additional details.
Parasite Viability Assay for qHTS and Follow-Up. Briefly, 3 μL of culture medium
was dispensed into 1,536-well black clear-bottom plates (Aurora Biotechnol-
ogies) using a Multidrop Combi (Thermo Fisher Scientific Inc.). Then, 23 nL
of compounds in DMSO were added by a pin tool (Kalypsys), and 5 μL of
P. falciparum-infected human red blood cells (0.3% parasitemia, 2.5% hema-
tocrit final concentration) were added. The plates were incubated at 37 °C in
a humidified incubator in 5% CO2 for 72 h, and 2 μL of lysis buffer (20 mM
Tris-HCl,10 mM EDTA, 0.16% saponin) weight to volume (w∕v), 1.6% Triton-X
(vol∕vol), 10× SYBR Green I (supplied as 10;000× concentration by Invitrogen)
were added to each well. The plates were mixed for 25 s with gentle shaking
and incubated overnight at 22–24 °C in the dark. The following morning,
fluorescence intensity at 485 nm excitation and 535 nm emission wavelengths
were measured on an EnVision (Perkin Elmer) plate reader. The compound
ACKNOWLEDGMENTS. We thank Dr. John Schwab [National Institute of
General Medical Sciences (NIGMS)] for his guidance of the CMLD Centers
Program, Dr. Paul Ralifo (BU) for assistance with NMR. Paul Shinn (NCGC)
for compound management, and Dr. Ruili Huang for informatics assistance
(NCGC). We also thank Dr. Paul Clemons (Broad Institute), Dr. Tanar Kaya
(Broad Institute), and Dr. J. Anthony Wilson (Broad Institute) for assistance
with principle moments of inertia analysis of the CMLD-BU compound collec-
tion. This work was generously supported by NIGMS CMLD Initiative (P50
GM067041 J.A.P., Jr.) and the Divisions of Intramural Research at the National
Institute of Allergy and Infectious Diseases and National Human Genome
Research Institute and the NIH Roadmap for Medical Research, all at the
National Institutes of Health.
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