Multi-Institution Research and Education Collaboration Identifies New Antimicrobial Compounds

Article abstract of DOI:10.1021/acschembio.0c00732

New antibiotics are urgently needed to address increasing rates of multidrug resistant infections. Seventy-six diversely functionalized compounds, comprising five structural scaffolds, were synthesized and tested for their ability to inhibit microbial growth. Twenty-six compounds showed activity in the primary phenotypic screen at the Community for Open Antimicrobial Drug Discovery (CO-ADD). Follow-up testing of active molecules confirmed that two unnatural dipeptides inhibit the growth of Cryptococcus neoformans with a minimum inhibitory concentration (MIC) ≤ 8 μg/mL. Syntheses were carried out by undergraduate students at five schools implementing Distributed Drug Discovery (D3) programs. This report showcases that a collaborative research and educational process is a powerful approach to discover new molecules inhibiting microbial growth. Educational gains for students engaged in this project are highlighted in parallel to the research advances. Aspects of D3 that contribute to its success, including an emphasis on reproducibility of procedures, are discussed to underscore the power of this approach to solve important research problems and to inform other coupled chemical biology research and teaching endeavors.

Full text of DOI:10.1021/acschembio.0c00732

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Articles
Multi-Institution Research and Education Collaboration Identifies
New Antimicrobial Compounds
Amelia A. Fuller,* Amy B. Dounay, Douglas Schirch, Daniel G. Rivera, Karl A. Hansford,
Alysha G. Elliott, Johannes Zuegg, Matthew A Cooper, Mark A. T. Blaskovich, Jacob R. Hitchens,
Sarah Burris-Hiday, Kristiana Tenorio, Yanira Mendez, J. Geno Samaritoni, Martin J. O’Donnell,
and William L. Scott*
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ABSTRACT: New antibiotics are urgently needed to address
increasing rates of multidrug resistant infections. Seventy-six
diversely functionalized compounds, comprising five structural
scaffolds, were synthesized and tested for their ability to inhibit
microbial growth. Twenty-six compounds showed activity in the
primary phenotypic screen at the Community for Open
Antimicrobial Drug Discovery (CO-ADD). Follow-up testing of
active molecules confirmed that two unnatural dipeptides inhibit
the growth of Cryptococcus neoformans with a minimum inhibitory
concentration (MIC) ≤ 8 μg/mL. Syntheses were carried out by
undergraduate students at five schools implementing Distributed
Drug Discovery (D3) programs. This report showcases that a
collaborative research and educational process is a powerful approach to discover new molecules inhibiting microbial growth.
Educational gains for students engaged in this project are highlighted in parallel to the research advances. Aspects of D3 that
contribute to its success, including an emphasis on reproducibility of procedures, are discussed to underscore the power of this
approach to solve important research problems and to inform other coupled chemical biology research and teaching endeavors.
he increasing rate of infections by multidrug resistant
T(MDR) microbes constitutes a serious threat to global
public health and economic output.1 MDR infections are a
consequence of overuse and misuse of existing antibiotics over
the past several decades coupled with the ability of bacteria and
fungi to rapidly develop varied resistance mechanisms.2−4
There is a critical need to prioritize the identification and
development of new therapeutic agents that can arrest and/or
prevent microbial growth, including compounds that employ
novel molecular mechanisms of action.
Phenotypic screening is an efficient approach to identify new
compounds that inhibit bacterial and fungal growth without
bias toward a specific molecular mechanism of action.5,6
Addressing the need for a reliable phenotypic screening
resource, the Community for Open Antimicrobial Drug
Discovery (CO-ADD) was launched in 2015 and has since
tested over 300,000 compounds.7−9 It affords free access to
assess the antimicrobial activity of submitted compounds (only
1 mg sample required) by testing their ability to inhibit growth
of “ESKAPE” pathogens: Escherichia coli, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, and methi-
cillin-resistant Staphylococcus aureus (MRSA), and the yeasts
Cryptococcus neoformans and Candida albicans. To leverage this
powerful resource to discover antimicrobial molecules, an
important synthetic need was identified: preparation of a large
set of compounds wherein varied structures and functionality
were prioritized.
Large numbers of molecules with diverse functionality are
readily and reproducibly available through the Distributed
Drug Discovery (D3) program implemented at a network of
global schools.10−15 Briefly, D3 enlists undergraduate students
enrolled in organic chemistry laboratory courses to prepare
combinatorial compound arrays for biological evaluation. To
date, D3 has successfully synthesized analogs of antimelanoma
compounds, for example.13 Indeed, a growing body of
literature reports significant research outcomes from curricu-
lum-based undergraduate research experiences (CUREs).16−23
In addition to providing a powerful method to achieve research
goals, CUREs, including D3, benefit students’ education.
CUREs deepen students’ understanding of scientific concepts,
Received: September 11, 2020
Accepted: November 19, 2020
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Figure 1. Schematic illustration of the parallel research process (blue) and education (tan) work flows in the D3/CO-ADD collaboration.
Figure 2. Structures of the five molecular scaffolds prepared and tested.
Figure 3. Bill-Board apparatus and an example combinatorial array. A) Picture of the Bill-Board equipment for parallel solid phase synthesis; B)
Example combinatorial array of scaffold 1 products. Phenylalanine (top row, green) or t-butyl-protected tyrosine (bottom row, blue) was N-
acylated with 4-, 3-, or 2-fluorobenzoic acid (left/violet, middle/gold, right/red columns, respectively).
enhance their confidence for future experiences, and equip
them to think independently.24,25 To meet the important
challenge of identifying new antimicrobial compounds, a D3/
CO-ADD collaboration integrates the critical need for new
antibiotics with the educational priorities of undergraduate
organic chemistry students (Figure 1).
simultaneously positively impacting undergraduate educational
experiences.
RESULTS AND DISCUSSION
■
Scaffold and Experimental Design Rationale. Access
to diverse, biomimetic molecules was prioritized in library
scaffold design. Each scaffold can be differently functionalized
at two or more sites, thereby providing access to a wide variety
of structures for screening. Recognizing that CO-ADD offered
a phenotypic screena successful tool in the discovery of
many new medicinesa specific molecular mechanism of
antimicrobial action was not targeted.5,6 Nonetheless, abun-
dant examples of bioactive molecules that contain each scaffold
exist, and expectations of antimicrobial activity were
reasonable. Marketed pharmaceuticals and bioactive analogs
that include these scaffolds have been comprehensively
surveyed,26 and a few examples were highlighted.
This article details the successful identification of antimicro-
bial compounds through a multi-institutional collaboration
involving D3 combinatorial library synthesis and CO-ADD
testing. Collaborators at five international institutions
IUPUI, Santa Clara University, Colorado College, Goshen
College, and the University of Havanasynthesized 76
functionally varied bio- and peptidomimetic molecules based
on five structurally unique scaffolds 1−5 (Figure 2). Of the 76
unique structures tested in a primary screen at CO-ADD, 26
compounds met a threshold level of antimicrobial growth
inhibition. Follow-up testing confirmed the ability of two
unnatural dipeptides 4 to inhibit the growth of Cryptococcus
neoformans with a minimum inhibitory concentration (MIC) ≤
8 μg/mL. Further, the successful identification of potent
antimicrobial compounds showcases the value of the D3
approach to discover new bioactive compounds while
The following drugs all contain N-acylated amino acid
motifs found in scaffolds 1 and 2: Alvimopan (GI surgery
recovery); L-DOPA (Parkinsonism); Folic Acid (Vitamin B9);
Lacosamide (epilepsy); Levothyroxine (hypothyroidism);
Liothyronine (hypothyroidism); Lymecycline (acne, some
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bacterial infections); Melphalan (multiple myeloma); Metho-
trexate (cancers); Mimosine (cancer); Pemetrexed (nonsmall
cell lung cancer); Pralatrexate (peripheral T-cell lymphoma);
Raltiterxed (malignant neoplasm).
Table 1. Varied Replication Location and Environments for
Scaffold Compounds
scaffold
replication location(s)
laboratory environment(s)
Scaffold 1 multisite replication
(Colorado College,
undergraduates engaged in
Like scaffold 3, these drugs all contained α-amino acid
amides: Atazanavir (HIV); Clindamycin (anaerobic bacteria);
Cobicistat (HIV); Valium (anxiety disorders); Lenalidomide
(multiple myeloma); Lidocaine (anesthesia); Lisdexamfet-
amine (ADHD); Lopinavir (HIV); Pomalidomide (multiple
myeloma); Ritonavir (HIV); Saxagliptin (type 2 diabetes
mellitus); Thalidomide (erythema nodosum leprosum).
For Scaffold 4 (dipeptides), bioactive examples included
Bortezomib (multiple myeloma); Ceftaroline (bacterial in-
fections); Enalapril and Lisinopril (hypertension); Penicillin V
(bacterial infections); Cialis (erectile dysfunction); Ximelaga-
tran (deep vein thrombosis). Additionally, there are anti-
microbial unnatural dipeptides that deliver their toxic payload
through the microbe’s specific dipeptide permease.27
independent faculty-led research;
large undergraduate laboratory
course; large scale workshop
University of Havana,
Goshen College, IUPUI)
Scaffold 2 internal replication
large scale undergraduate laboratory
course
(IUPUI)
Scaffold 3 internal replication
large scale undergraduate laboratory
course
(IUPUI)
Scaffold 4 internal replication
undergraduates in independent
faculty-led research group
(IUPUI)
Scaffold 5 bilateral replication (Santa
undergraduates in independent
faculty-led research group
Clara University, IUPUI)
impacts: it strengthened synthetic and biological results and
offered a robust training opportunity for students just learning
the research process.
A bioactive arylopeptoid28−30 (Scaffold 5 analog) was
reported.31 Further, the N-substituents in the oligo-amide
backbone of arylopeptoid scaffold 5 are analogous to those
found in peptoids; many examples of anti-infective peptoids
have been reported.32
The inclusion of a “control” reaction in the six-reaction array
represented a second way in which reproducibility and
experimental design were emphasized. Successful synthesis of
the control demonstrated students’ mastery of the synthetic
techniques applied to the preparation of the five new
compounds. Notably, the fate of the new syntheses was not
predetermined, an aspect of authentic research. When controls
gave the expected result but a new synthesis did not, the
unexpected observation, if replicated, became an incentive to
propose alternative explanations rather than dismiss it as due
to “failure of technique”. Although we do not report any
examples that follow this path in this work, this experimental
design feature is important to teach students that properly
conducted “unsuccessful” experiments has previously led to
hypotheses, further research, and discoveries.37
Library Synthesis. Scaffold 1. Examples of the simplest
scaffold, the N-acylated natural amino acids (Scheme 1, 1.1−
1.27), comprised diversity from the commercially available
combinatorial reagents, Fmoc-amino acid-functionalized Wang
resins and carboxylic acids. The 27 analogs were prepared in
three synthetic steps: (1) removing the Fmoc protecting group
from the amino acid-functionalized resin, (2) coupling to a
carboxylic acid, and (3) liberating the product from the resin
with concomitant removal of amino acid side-chain protection
(Scheme 1). To access structures 1.1−1.27, either phenyl-
alanine, tyrosine, or isoleucine was acylated with benzoic acid
or a substituted benzoic acid derivative. Because of the brevity
of the procedure and the availability of inexpensive
combinatorial reagents, a large number of student researchers
prepared variants of 1 (Table 1).15
Scaffolds 2−4 via Intermediate A. Three scaffoldsN-
acylated unnatural amino acids 2 (2.1−2.12), unnatural α-
amino acid amides 3 (3.1−3.12), and unnatural dipeptides 4
(4.1−4.13) that contain an unnatural amino acidwere
prepared from a common, racemic, Wang resin-bound
unnatural amino acid intermediate, A (Scheme 2). Con-
sequently, all the final products 2-4 were either racemic or a
mixture of diastereomers. Screening of limited stereochemical
mixtures was considered an advantage in preliminary
identification of bioactive molecules.
To access intermediate A, a resin bearing the benzophenone
imine of glycine was alkylated.38−40 This step introduced
considerable structural diversity from a rich pool of alkyl or
benzyl halides, and stereoisomeric mixtures were formed.
D3 methods were identified as an efficient approach to
prepare an initial screening library of widely varied analogs
within each scaffold. The D3 strategy involved students
enrolled in independent or course-based research experiences
executing straightforward, highly reproducible combinatorial
procedures on solid support. In the hands of novice student
researchers, reactions on solid support are highly successful
owing to the introduction of reagents in large excess, simple
workups, and the absence of purification steps for synthetic
intermediates. Meeting the need for large numbers of
compounds, individual students or student teams synthesized
arrays of six compounds using the “Bill-Board” apparatus
(Figure 3A). The Bill-Board is a commercially available,
affordable, and compact array of six solid-phase reaction
vessels.10 Figure 3B shows an example combinatorial array that
was prepared by a student group using this equipment.
The participation of several institutions and scientists was
leveraged to enhance the number and diversity of examples
prepared as well as the educational experiences of the students
involved in these projects. Because D3 engaged many
researchers at a variety of institutions, collaborators selected
the scaffold chemistry most compatible with their school’s
resources, expertise, and skill level. Simultaneously, students
were highly motivated by the opportunity to contribute to
finding solutions to an important societal problem while
learning essential organic chemistry concepts and experimental
techniques.
Reproducibility, a fundamental science requirement, was
prioritized in the synthesis and evaluation of the initial
screening libraries. Despite its recognized importance, how and
when reproducibility is demonstrated is the subject of much
discussion and controversy.33−36 We demonstrated in this
collaboration that meaningful replication goes well beyond a
single researcher and his or her own experimental results.
Except for four structures (1.5, 1.27, 4.12, and 4.13), each of
the compounds was synthesized in replicate in one or more of
the varied environments shown in Table 1, always by two or
more scientists, often at different levels of expertise, and
sometimes at different global locations. An emphasis on
meaningful replication in these experiments had tandem
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a
Scheme 1. Synthesis of 1.1−1.27
a
Reaction times were varied to match instructional laboratory schedules, as detailed in the methods. NMP: N-methyl-2-pyrrolidone, DIC: N,N′-
diisopropylcarbodiimide, HOBt: hydroxybenzotriazole, TFA: trifluoroacetic acid.
a
Scheme 2. Synthesis of 2.1−2.12, 3.1−3.12, and 4.1−4.13 via Intermediate A
a
Reaction times were varied to match instructional laboratory schedules, as detailed in the methods. THF: tetrahydrofuran, MeOH: methanol,
NMP: N-methyl-2-pyrrolidone, DIC: N,N′-diisopropylcarbodiimide, DIEA: N,N′-diisopropylethylamine, HOBt: hydroxybenzotriazole, TFA:
trifluoroacetic acid.
Acidic hydrolysis of the imine provided the key resin-bound,
racemic, unnatural amino acid intermediate A.38,39
Scaffold 2. N-acylated compounds 2.1−2.12 were prepared
by DIC/HOBt-mediated reaction of 12 substituted phenyl-
alanine analogs of A with one of four substituted benzoic or
heteroaromatic carboxylic acids, followed by acidic cleavage
from the resin.
Scaffold 3. Twelve substituted phenylalanine amide analogs
(3.1−3.12) were prepared directly from aminolytic cleavage of
the resin link in A with ammonia or methylamine.
Scaffold 4. Unnatural dipeptides 4.1−4.13 were accessed by
acylating two substituted fluorophenyalanine analogs A with
seven different BOC-protected amino acids. This was followed
by acidic cleavage from the resin with concomitant N- and side
chain functionality deprotection.
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a
Scheme 3. Synthesis of 5.1−5.12
a
DIEA: N,N′-diisopropylethylamine, DMSO: dimethylsulfoxide, DMF: N,N-dimethylformamide.
Scaffold 5. To prepare the final scaffold, the arylopeptoids
(Scheme 3, 5.1−5.12),28−30 a chlorotrityl chloride resin was
functionalized with 3-(chloromethyl)benzoic acid, and the
primary chloride was subsequently displaced by one of six
primary amines. The resultant secondary amine was acylated
with 3-(chloromethyl)benzoyl chloride. Again the primary
chloride was displaced by one of four primary amines, and the
N-terminus was capped with acetic anhydride before cleaving
the molecule from the resin under acidic conditions.30
Arylopeptoids 5.1−5.12 derived structural diversity from the
alkyl, heterocyclic, and aromatic primary amines incorporated.
Synthesis summary. For each scaffold, at least 12
representative compounds were prepared in duplicate or
quadruplicate (with the exception of 1.5, 1.27, 4.12 and
4.13, which were prepared only once). These replications were
always by separate researchers. Crude purities (typically >85%,
Supplementary Table S2) and identities of products were
assessed by liquid chromatography−mass spectrometry (LC-
MS). These crude products were suitable for biological
evaluation without further purification. Select compounds
were purified by chromatography, most commonly because the
curriculum plan included a purification step.
structure−activity relationships for molecules that exhibited
biological activity.
Biological evaluation. All compounds synthesized were
submitted to CO-ADD for evaluation of their ability to inhibit
the growth of five bacteria (Escherichia coli, Klebsiella
pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa
and Staphylococcus aureus), and two yeasts (Candida albicans
and Cryptococcus neoformans). The complete set of results is in
Supplementary Table S2. Impressively, 26 of the 76 unique
molecules inhibited growth of one or more microbes in the
primary screen at 32 μg/mL. Structures of all molecules with
biological activity are shown in Figure 4. Seventeen unique
molecules were identified as being “partially active” (i.e., they
inhibited 50−80% of growth of one or more pathogens in the
assay, Figure 4A). The partially active molecules comprised
examples from all five scaffolds, suggesting that any of these is a
promising template for further synthesis and evaluation. Five
compounds showed partial activity replicated across duplicate
lots (2.8, 3.2, 4.8, 4.11, and 5.4). Nine unique structures were
identified as “active” (i.e., they inhibited >80% of pathogen
growth, Figure 4B), and the activity of five of these (3.10, 4.1,
4.4, 4.7, 4.9) was replicated in both sets of duplicate
compounds. The active molecules include three structural
scaffolds: N-acylated natural amino acids (1.6 and 1.12), an
unnatural α-amino acid amide (3.10), and unnatural
dipeptides (4.1, 4.4, 4.7, 4.9, 4.12, 4.13).
The easy synthetic access to a diverse set of analogs,
showcased by the 76 compounds prepared here, enhances the
value of these scaffolds as a continuing source of potential drug
candidates. The procedures for their preparation are rigorously
validated and accessible to chemists working in varied
laboratory conditions,12 enabling ready design and synthesis
of new analogs. Future synthetic targets will explore key
Microbial growth inhibition was not always reproduced
between two lots of the same compound. For example, the
inhibition of the growth of three organisms observed for one
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Figure 4. Structures of molecules with activity in the biological screens at CO-ADD. The pathogen inhibited is indicated below each compound.
*The compound showed replicated biological activity when duplicate compounds were tested. A) Structures that inhibited 50−80% of pathogen
growth (“partially active”) in the primary screen at 32 μg/mL; B) Structures that inhibited >80% of pathogen growth (“active”) in the primary
screen. Structures 4.1 and 4.4 in the red box were active in preliminary screen, with subseqently confirmed MIC values and no cytotoxicity at the
highest concentrations tested in the secondary hit confirmation screen.
lot of 1.12 was not replicated in the other three lots. There are
serveral possible sources for these inconsistencies, including
differences in actual concentrations and/or purity of the two or
more lots of compounds submitted, errors in compound
transfers, and biological assay variability. The specific causes of
nonreplicated activity across duplicate lots were not pursued.
Nonetheless, the variability between sets of duplicate
compounds reiterated that replication at all stages of the
research process is essential; decisions are not based on a single
observation or experiment.
Each lot of the 26 compounds that showed at least partial
activity in the preliminary screening was subsequently
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Bill-Board was allowed to stand stationary after manual inversion of
the Bill-Board a few times to effect mixing. Additionally, two
procedures for cleavage of the reaction products from the resin have
been used effectively and interchangeably. Sources of materials as well
as the preparation of the benzophenone imine of glycine-Wang resin
used for the synthesis of intermediate A are detailed in the Supporting
Information. Structures, purities, masses, and school source for all
replicates of all compounds prepared are tabulated in Supporting
Information Table S2.
Synthesis of N-Acylated Natural Amino Acids (1.1−1.26). These
compounds were prepared according to previously reported
methods,15 which are further detailed in the Supporting Information.
Synthesis of Unnatural Amino Acid Intermediate A. Intermediate
A was prepared as previously reported.10,12 Details of the synthesis are
included in the Supporting Information.
Synthesis of N-Acylated Unnatural Amino Acids (2.1−2.12).
These compounds were prepared according to previously reported
methods;10 these are further detailed in the Supporting Information.
Synthesis of α-Amino Acid Amides (3.1−3.12). From inter-
mediate A, reaction vessels were washed 3 × 2 mL with THF, and
reaction vessel bottom caps were replaced. Either ammonia (2.5 mL
of 7 N solution in methanol) or methylamine (2.5 mL of a 33%
solution in ethanol) were added. The Bill-Board was capped and
inverted three times, then allowed to sit at RT for 12 days. The
reaction vessel caps were then removed, and the solution was
collected into individually labeled, tared vials. Each reaction vessel was
rinsed with an additional 2 × 2 mL with THF. A small aliquot of the
filtrate (100 μL) was evaporated to dryness for LC/MS analysis. The
remainder was concentrated by evaporation under a stream of N2.
Synthesis of Dipeptides (4.1−4.13). From intermediate A, the
reaction vessels were capped at the bottoms. A solution was prepared
of the appropriate acid (0.25 M solution in 0.25 M HOBt in NMP).
To each reaction vessel, 1 mL of the appropriate solution was added
(0.25 mmol acid, 5 equiv acid, 0.25 mmol HOBt, 5 equiv HOBt)
followed by 0.5 mL of DIC solution (0.5 M in NMP, 0.25 mmol, 5
equiv). The tops of the reaction vessels were capped, and the Bill-
Board was allowed to rotate for 2−5 days. The reaction vessels were
uncapped, and each was washed twice with NMP, twice with THF,
and thrice with CH2Cl2. Reaction vessels were capped at the bottom,
then each was treated with 2 mL of a mixture of TFA/CH2Cl2/H2O
for 30 min. The reaction vessel caps were then removed, and the
solution was collected into individually labeled, tared vials. Each
reaction vessel was rinsed with an additional 2 mL of the cleavage
solution, then 2 mL CH2Cl2. A small aliquot of the filtrate (100 μL)
was evaporated to dryness and was analyzed by LC/MS. The
remainder was concentrated by evaporation under a stream of N2
(effluent TFA vapor was scrubbed in a caustic solution of sodium
hydroxide).
subjected to follow-up hit confirmation screening, with MICs
for each microbe determined contingent upon verification of
activity. Compounds were also tested for cytotoxicity to a
mammalian cell line (HEK293, human embryonic kidney).
From this more stringent assay, two unnatural dipeptides (4.1,
4.4, Figure 4B) were confirmed to inhibit growth of C.
neoformans with an MIC ≤ 8 μg/mL. This value is comparable
to the MIC against this fungus measured for the common
antifungal fluconazole under identical screening conditions.41
It was encouraging that neither of these unnatural dipeptides
inhibited the viability of HEK293 cells at the highest
concentrations tested (32 μg/mL) (see Supporting Informa-
tion). The relatively low number of molecules that emerged as
confirmed hits underscores the importance of applying this
rigorous two-step screening process to identify new bioactive
molecules.
In this work, we have demonstrated that the D3/CO-ADD
collaboration is a powerful strategy to couple combinatorial
synthesis and phenotypic screening to identify new antimicro-
bial compounds. The identification of two readily accessible
molecules that inhibit Cryptococcus neoformans growth (4.1 and
4.4), verified over two screening steps (preliminary and hit
confirmation), is an exciting initial accomplishment. A more
detailed investigation to validate and evaluate the scope of the
antifungal activity of 4.1, 4.4 and analogs, as single
diastereomers, is underway. Further, the successful preparation
of 76 molecules comprising five different scaffolds highlights
the reliability of the procedures developed. In parallel, student
researchers were equipped with valuable hands-on learning
through the D3 processes.
Based on these experiences and previous reports,11,15,28 we
offer our perspectives on important factors for the success,
sustainability, and future of the D3/CO-ADD collaboration.
One key to sustained success is that D3/CO-ADD experiments
meet the resources and needs of particular institutions (e.g.,
number of students, costs, available instrumentation). They
employ cost-effective, operationally straightforward equipment,
like the Bill-Board42,43 to prepare numerous and diverse
analogs. In doing so, both the clarity of procedures and
robustness of chemistry are validated. Finally, we find that this
project provides a meaningful context for learning basic
organic chemistry experimental techniques and theory. The
D3/CO-ADD collaboration model is well-positioned to be a
template for other investigators who may adapt these strategies
to design new scaffolds for synthesis and subsequent biological
evaluation, thereby advancing the pace of discovery of new
antimicrobial compounds.
Synthesis of Arylopeptoids (5.1−5.12). The preparation of these
was carried out according to previously reported methods;28 these are
further detailed in the Supporting Information.
LC/MS Analysis of Synthetic Products. Molecules prepared were
characterized exclusively by LC/MS to evaluate purity and identity
(methods detailed in the Supporting Information). Crude purities of
synthesized products were determined by integration of the
chromatograms (Table S2).
Preparation of Samples for Submission to CO-ADD. Solutions of
samples were prepared in known volumes of either methanol
(scaffolds 2−4) or acetone (scaffolds 1 and 5). An aliquot
representing 1.0 mg was then transferred to a barcoded vial provided
by CO-ADD and the solvent was removed by evaporation using a
stream of nitrogen gas.
METHODS
■
Chemistry. Safety Note. DIC is a known irritant and contact
sensitizer.44 Care with any peptide coupling reagents is advised owing
to their documented health hazards.45
General Notes. The general procedures documented below for the
syntheses of Scaffolds 2 and 5 compounds were performed using the
methods previously described.10−13,28,38 The Bill-Board apparatus42
was used for the synthesis of all scaffolds. The utility and accessibility
of this equipment has been previously described in detail.10−12,15,28,46
Slight modifications to general procedures were implemented in
different laboratory environments, and reactions outcomes were
comparable despite these changes. Reaction times were varied to
accommodate the frequency of laboratory meetings for students
enrolled in organic chemistry classes. Reactions were performed in
some laboratories with agitation provided by a motor-driven rotation
assembly. However, reaction outcomes were not compromised if the
Biology. All preliminary screening and hit confirmation assay
results are tabulated in Table S2.
Preliminary Screening Sample Preparation. Samples received by
CO-ADD were stored frozen at −20 °C. Samples were prepared in
DMSO and water to a final testing concentration of 32 μg/mL or 20
μM in 384-well, nonbinding surface plate (NBS) for each bacterial/
fungal strain, and in duplicate (n = 2), and keeping the final DMSO
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concentration to a maximum of 1% DMSO. All the sample
preparation solution transfers were done using liquid handling robots.
Preliminary Screening Assays. Preliminary antibacterial and
antifungal screening assays were carried out following reported
procedures.9 Additional details are in the Supporting Information.
Preliminary Screening Analysis of Assay Results. Percentage
growth inhibition was calculated for each treated well using the
absorbance readouts, in comparison with median absorbance value of
untreated bacteria (positive growth control) and with median
absorbance value of media only (negative growth control):
AUTHOR INFORMATION
Corresponding Authors
■
Amelia A. Fuller − Santa Clara University, Department of
Chemistry & Biochemistry, Santa Clara, California 95053,
United States; orcid.org/0000-0002-9291-6037;
Email: aafuller@scu.edu
William L. Scott − Department of Chemistry and Chemical
Biology, Indiana University Purdue University Indianapolis,
Indianapolis, Indiana 46202, United States; Email: wscott@
iupui.edu
OD − median(ODNegControl
median(ODPosControl) − median(ODNegControl
)
i
inhibitioni = 1 −
MScorei =
)
Authors
Amy B. Dounay − Department of Chemistry and
Biochemistry, Colorado College, Colorado Springs, Colorado
80903, United States; orcid.org/0000-0003-0451-5549
Douglas Schirch − Department of Chemistry, Goshen College,
Goshen, Indiana 46526, United States
Daniel G. Rivera − Center for Natural Products Research,
Faculty of Chemistry, University of Havana, 10400 La
Habana, Cuba; orcid.org/0000-0002-5538-1555
Karl A. Hansford − Community for Open Antimicrobial Drug
Discovery, Centre for Superbug Solutions, Institute for
Molecular Bioscience, The University of Queensland, St.
Lucia, Queensland 4072, Australia; orcid.org/0000-
0003-2535-9071
Alysha G. Elliott − Community for Open Antimicrobial Drug
Discovery, Centre for Superbug Solutions, Institute for
Molecular Bioscience, The University of Queensland, St.
Lucia, Queensland 4072, Australia; orcid.org/0000-
0002-2983-0484
Johannes Zuegg − Community for Open Antimicrobial Drug
Discovery, Centre for Superbug Solutions, Institute for
Molecular Bioscience, The University of Queensland, St.
Lucia, Queensland 4072, Australia
Matthew A Cooper − Community for Open Antimicrobial
Drug Discovery, Centre for Superbug Solutions, Institute for
Molecular Bioscience, The University of Queensland, St.
Lucia, Queensland 4072, Australia; orcid.org/0000-
0003-3147-3460
Mark A. T. Blaskovich − Community for Open Antimicrobial
Drug Discovery, Centre for Superbug Solutions, Institute for
Molecular Bioscience, The University of Queensland, St.
Lucia, Queensland 4072, Australia
Jacob R. Hitchens − Department of Chemistry and Chemical
Biology, Indiana University Purdue University Indianapolis,
Indianapolis, Indiana 46202, United States
Sarah Burris-Hiday − Department of Chemistry and Chemical
Biology, Indiana University Purdue University Indianapolis,
Indianapolis, Indiana 46202, United States
ˆ
0.6745(OD − OD)
i
MAD
The significance of the inhibition values was determined by modified
Z-scores, calculated using the median and MAD of the samples (no
controls) on the same plate. In the preliminary screening, samples
with inhibition value above 80% and Z-Score above 2.5 for either
replicate (n = 2 on different plates) were classed as actives. Samples
with inhibition values between 50−80% and Z-Score above 2.5 for
either replicate (n = 2 on different plates) were classed as partial
actives.
Preliminary Screening, Antibiotic Standards Preparation, and
Quality Control. Colistin and Vancomycin were used as positive
bacterial inhibitor standards for Gram-negative and Gram-positive
bacteria, respectively. Fluconazole was used as a positive fungal
inhibitor standard for C. albicans and C. neoformans. The antibiotics
were provided in four concentrations, with 2 above and 2 below their
MIC values, and plated into the first eight wells of column 23 of the
384-well NBS plates. The quality control (QC) of the assays was
determined by the antimicrobial controls and the Z′-factor (using
positive and negative controls). Each plate was deemed to fulfill the
quality criteria (pass QC), if the Z′-factor was above 0.4, and the
antimicrobial standards showed full range of activity, with full growth
inhibition at their highest concentration, and no growth inhibition at
their lowest concentration.
Hit Confirmation Screening, MIC Determination, and Cytotox-
icity Assay. Compounds identified as partially active or active in the
preliminary screen were subjected to hit confirmation screening, MIC
determination, and cytotoxicity assays following reported methods.9
Additional details are provided in the Supporting Information.
Hit Confirmation Quality Control. All screenings were done as two
replica (n = 2), with both replicas on two different plates, but from
single plating and done in a single screening (microbial incubation).
In addition, two values are used as quality control for individual
plates:
3 × (MAD(ODPosControl) + MAD(ODNegControl))
|median(ODPosControl) − median(ODNegControl)|
and standard antibiotic controls at different concentrations (above
and below their MIC). The plate passes the quality control if Z′-
Factor >0.4 and standards are active and inactive at highest and lowest
concentrations, respectively.
Kristiana Tenorio − Santa Clara University, Department of
Chemistry & Biochemistry, Santa Clara, California 95053,
United States
Yanira Mendez − Center for Natural Products Research,
Faculty of Chemistry, University of Havana, 10400 La
Habana, Cuba; orcid.org/0000-0003-2124-4912
J. Geno Samaritoni − Department of Chemistry and Chemical
Biology, Indiana University Purdue University Indianapolis,
Indianapolis, Indiana 46202, United States
Martin J. O’Donnell − Department of Chemistry and
Chemical Biology, Indiana University Purdue University
Indianapolis, Indianapolis, Indiana 46202, United States
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschembio.0c00732.
■
sı
Synthetic procedures (21 pages) for preparation of
analogs of previously reported scaffolds 1, 2, and 5;
tabulated conditions for LC/MS analyses of synthetic
products; previously reported biological screening
methods; tabulated synthetic and biological screening
data for all compounds reported (PDF)
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschembio.0c00732
H
https://dx.doi.org/10.1021/acschembio.0c00732
ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology
pubs.acs.org/acschemicalbiology
Articles
Unnatural Amino Acids and Virtual D3 Catalog Construction. J.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank the following agencies for financial support: National
Science Foundation (CHE-1056520 and CHE-1566604,
awarded to A.A.F., DUE-1140602 awarded to M.J.O. and
W.L.S.), National Institutes of Health (R01-GM28193,
awarded to M.J.O.). Antimicrobial and cytotoxicity screening
was performed by CO-ADD (The Community for Antimicro-
bial Drug Discovery), funded by the Wellcome Trust Strategic
Award 104797/Z.14/Z (UK) and The University of Queens-
land (Australia). M.J.O., W.L.S., and J.G.S. thank M.
Schonberger of Lilly Analytical Technologies for analytical
support. A.B.D. thanks T. Cervantes, R. Woncier, and T. T.
Pham for analytical and technical support at Colorado College.
We thank P. Dave, J. Sanchez, and D. Tiano for their assistance
as TAs in the Havana laboratories, and the large number of
student researchers who were enrolled in organic chemistry
laboratory courses and carried out D3 experiments, including
those described here: SCU, 14 students; CC, 144 students;
GC, 8 students; UH, 27 students; IUPUI, 277 students.
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