Design, synthesis, and evaluation of substituted nicotinamide adenine dinucleotide (NAD+) synthetase inhibitors as potential antitubercular agents

Article abstract of DOI:10.1016/j.bmcl.2017.08.012

Nicotinamide adenine dinucleotide (NAD⁺) synthetase catalyzes the last step in NAD⁺ biosynthesis. Depletion of NAD⁺ is bactericidal for both active and dormant Mycobacterium tuberculosis (Mtb). By inhibiting NAD⁺ synthetase (NadE) from Mtb, we expect to eliminate NAD⁺ production which will result in cell death in both growing and nonreplicating Mtb. NadE inhibitors have been investigated against various pathogens, but few have been tested against Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied to understand the structure–activity-relationships of substituents on this position. Compounds were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent compound displayed very weak inhibition against Mtb NadE (IC₅₀ = 1000 μM), we observed up to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of the series with an IC₅₀ value of 90 μM against Mtb NadE. Our modeling results show that these urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is supported by data showing that, even when treated with potent inhibitors, NadE catalysis is restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb cell growth with MIC values of 19–100 μg/mL. These results improve our understanding of the SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a potential antitubercular drug target.

Full text of DOI:10.1016/j.bmcl.2017.08.012

Accepted Manuscript
Design, synthesis, and evaluation of substituted nicotinamide adenine dinucleo-
tide (NAD+) synthetase inhibitors as potential antitubercular agents
Xu Wang, Yong-Mo Ahn, Adam G. Lentscher, Julia S. Lister, Robert C.
Brothers, Malea M. Kneen, Barbara Gerratana, Helena I. Boshoff, Cynthia S.
Dowd
PII:
S0960-894X(17)30800-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.012
BMCL 25206
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 June 2017
5 August 2017
7 August 2017
Please cite this article as: Wang, X., Ahn, Y-M., Lentscher, A.G., Lister, J.S., Brothers, R.C., Kneen, M.M.,
Gerratana, B., Boshoff, H.I., Dowd, C.S., Design, synthesis, and evaluation of substituted nicotinamide adenine
dinucleotide (NAD+) synthetase inhibitors as potential antitubercular agents, Bioorganic & Medicinal Chemistry
Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.08.012
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Design, synthesis, and evaluation of substituted nicotinamide adenine dinucleotide (NAD+)
synthetase inhibitors as potential antitubercular agents
Xu Wang1, Yong-Mo Ahn2, Adam G. Lentscher1, Julia S. Lister1, Robert C. Brothers1, Malea M.
Kneen3,a, Barbara Gerratana4, Helena I. Boshoff2, Cynthia S. Dowd1*
1Department of Chemistry, George Washington University, Washington DC 20052
2Tuberculosis Research Section, LCID, NIAID/NIH, Bethesda, MD 20892
3Department of Chemistry and Chemical Biology, Indiana University—Purdue University
Indianapolis, Indianapolis, IN 46202
4Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742
*Corresponding author
800 22nd Street NW, Suite 4000
Washington DC 20052
+1-202-994-8405 (ph)
cdowd@gwu.edu
Present Address:
aRoche Diagnostics Corporation, Indianapolis, IN 46250
Keywords: Mycobacterium tuberculosis, NadE, antibiotic, antitubercular

Abstract
Nicotinamide adenine dinucleotide (NAD+) synthetase catalyzes the last step in NAD+
biosynthesis. Depletion of NAD+ is bactericidal for both active and dormant Mycobacterium
tuberculosis (Mtb). By inhibiting NAD+ synthetase (NadE) from Mtb, we expect to eliminate
NAD+ production which will result in cell death in both growing and nonreplicating Mtb. NadE
inhibitors have been investigated against various pathogens, but few have been tested against
Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by
Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied
to understand the structure-activity-relationships of substituents on this position. Compounds
were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent
compound displayed very weak inhibition against Mtb NadE (IC50 = 1000 µM), we observed up
to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group
on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of
the series with an IC50 value of 90 µM against Mtb NadE. Our modeling results show that these
urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports
ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is
supported by data showing that, even when treated with potent inhibitors, NadE catalysis is
restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb
cell growth with MIC values of 19-100 µg/mL. These results improve our understanding of the
SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a
potential antitubercular drug target.

Tuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (Mtb), remains
one of the world's deadliest infectious diseases.1-3 According to the World Health Organization
(WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS), 10.4 million people
fell ill and 1.8 million died from TB in 2015, which is 0.7 million more than those who died from
HIV-related illnesses.1, 2 Besides the high prevalence of TB, the large number of new cases of
multi-drug resistant (MDR) and extensively-drug resistant (XDR) TB has made the disease a
more serious public health concern.2 Two of the most important first-line TB drugs (isoniazid,
rifampicin) are both ineffective against MDR-TB and XDR-TB, rendering the treatment options
very limited.4, 5 Thus, there remains a pressing need for novel drugs that shorten TB treatment
and are effective against all pathogenic strains.
Nicotinamide adenine dinucleotide (NAD+) is a ubiquitous enzyme cofactor,
indispensable for reduction-oxidation reactions as well as essential nonredox functions in the cell
such as cell longevity, telomere maintenance, Ca2+ signaling, DNA repair, and immune
response.6, 7 NAD+ synthetase (NadE) is an essential enzyme that catalyzes the last step in many
NAD+ de novo biosynthesis and NAD+ recycling pathways.8, 9 In Mtb, NadE transforms nicotinic
acid adenine dinucleotide (NaAD+) into NAD+ via a two-step process with the assistance of ATP
and ammonia (Figure 1).8-13 Ammonia is obtained from glutamine hydrolysis in the glutaminase
domain of the enzyme.8-13 Inhibition of NadE blocks NAD+ biosynthesis and leads to cell death
in both growing and nonreplicating Mtb.14-16 The importance of NAD+ encourages the design of
NadE inhibitors that may be effective against both active and latent tuberculosis. Moreover, the
low sequence identity of 23% between Mtb NadE and the human homolog, as well as the

presence of NadE-independent NAD+ biosynthesis pathways in humans, increases the attraction
of NadE as a drug target for Mtb.7, 13, 17, 18
Figure 1. Two-step reaction catalyzed by NAD+ synthetase.
Despite this promise, few studies explore NadE inhibitors as antitubercular agents. Velu
et al. reported a series of tethered dimers as inhibitors of B. subtilis NadE and several
Gram-positive organisms.16, 19 One of the most potent B. subtilis NadE inhibitors from this work
(Figure 2A) yielded an IC50 (concentration resulting in 50% enzyme inhibition) value of 10 µM
against B. subtilis NadE and an MIC (minimum inhibitory concentration) of 1.5 µM against B.
subtilis.16, 19 Boshoff et al. tested several of these tethered dimers against Mtb NadE and Mtb
cellular growth.14 The compounds, however, showed only modest activity. The most potent Mtb
NadE inhibitor (Figure 2B) gave an IC50 of 21.8 µM against Mtb NadE and an Mtb MIC of 20
µM.14

BnO
O
O
N
N
N
N
7 O
7 O
CO2Me
CO2Me
A
B
B. subtilis NadE IC50 = 10 µM
B. subtilis MIC = 1.5 µM
Mtb NadE IC50 = 21.8 µM
Mtb MIC = 20 µM
H
H
H
H
N
N
NO2
N
N
O
O
H
N
Cl
Cl
O
S
O
S
NO2
N
H
O
O
H2NH2C
5824
B. anthracis NadE IC50 = 6.4 µM
C
B. anthracis NadE IC50 = 15.3 µM
µ
B. anthracis MIC > 240
M
Figure 2. Structures and bioactivities of known NadE inhibitors.14, 16, 19, 20
A significant number of compounds as inhibitors of NadE have been synthesized by the
Brouillette group.20, 21 Using a high-throughput assay, they discovered compound 5824, which
showed strong inhibition of B. anthracis NadE (IC50 = 6.4 µM, Figure 2).20 According to their
modeling results using the B. subtilis homolog, the group predicted that 5824 bound to the
NaAD+ subsite of NadE.20 The group next reported a series of the reverse sulfonamide analogs of
5824 that were tested against B. anthracis NadE, B. anthracis NaMNAT, and B. anthracis Sterne.
One of their best inhibitors (Figure 2C) displayed a B. anthracis NadE IC50 of 15.3 µM but did
not kill B. anthracis cells.21
In order to evaluate compounds that are active against Bacillus NadE as potential Mtb
NadE inhibitors, the similarity between Bacillus and Mtb homologs was explored. Mtb NadE is
glutamine dependent and has a glutaminase domain that transports ammonia to the synthetase
domain via an ammonia tunnel.11 Interestingly, NadE from B. subtilis or B. anthracis depends on

exogenous ammonia and does not possess a glutaminase domain or an ammonia tunnel.22, 23 Thus,
the amino acid sequences of NadE from B. subtilis, B. anthracis, and only the C-terminal domain
of Mtb NadE (i.e., the Mtb NadE synthetase domain that is homologous to the Bacillus NadE
enzymes) were aligned. The sequence identity among these enzymes was calculated based on
this alignment using MUSCLE24, 25 (Table 1). While the two Bacillus NadEs share 88.6%
sequence identity, the Mtb NadE C-terminal domain shares 36.6% sequence identity to the
Bacillus subtilis NadE and 34.4% sequence identity to the Bacillus anthracis NadE. We expected
high conservation of the active site residues between species, which encourages the design of
Mtb NadE inhibitors based on the Bacillus inhibitor structures. Therefore, we chose compound
5824 (3-{4-[(3,4-dichlorophenyl)sulfamoyl]phenyl}-1-(4-nitrophenyl)urea, Figure 2) as the
parent structure for the current work.
Table 1. Sequence identity between NadE synthetase homologs from Mtb, B. subtilis and B.
anthracis
Mtb NadE
B. subtilis
B. anthracis
(C-term
Synthetase
domain)
Sequence Identity (%)
NadE
NadE
Mtb NadE C-term Synthetase domain
B. subtilis NadE
36.6
88.6
B. anthracis NadE
34.4

A virtual library of 118 urea-sulfonamide analogs was made. Half of the compounds were
sulfonamides, retaining the configuration of parent compound 5824, while half were the
‘reversed sulfonamide’, corresponding to the opposite configuration. Compounds varied
structurally only at ring A, where a variety of substituents were appended. Substituents were
selected based on the Topliss approach toward aromatic systems26 as well as commercially
available anilines. Compounds were docked into the crystal structure of Mtb NadE13 (PDB id:
3DLA) using the Glide tool in Schrödinger, suite 201027. The 100 top-scoring analogs are shown
in Table S1 (more negative Glide scores predicting improved binding affinities). Interestingly,
the putative binding site of many analogs was predicted to involve the intramolecular ammonia
tunnel11 that transports ammonia from the glutaminase domain to the synthetase domain (Table
S2). For example, compound 4l (R = 4-H) formed hydrogen bonds with GLY-366 and LEU-399
(Figure 3A), and ring A rested in a hydrophobic pocket, the entrance to the tunnel (Figure 3B).
Based on these modeling results and synthetic feasibility, a series of analogs was selected for
synthesis (Figure 4). These compounds could shed light on the influence of ring A structural
changes on Mtb NadE inhibition and antibacterial activity.

Figure 3. Compound 4l (R = 4-H) docked into Mtb NadE (PDB: 3DLA). (A) The green ribbon represents NadE.
The ligand and selected protein residues are colored by atom type, and hydrogen bonding is represented by
dashed red lines. (B) Using the same NadE orientation, the surface of the enzyme is colored to show the degree
of hydrophobicity (darker red indicates more hydrophobic character). Ring A (circled) is shown going into the
ammonia tunnel at its entrance.
Figure 3. Structure of parent compound 5824 and the analogs prepared in this work.
To prepare the desired compounds, a simple synthetic route was designed (Scheme 1).
Substituted anilines 1a-l were combined with 4-acetamidobenzenesulfonyl chloride to obtain
compounds 2a-l generally in high yield unless a second recrystallization in ethanol was needed.
The deacetylated products (3a-l) were obtained after acid hydrolysis and were purified either by
silica column chromatography or recrystallization. The intermediates 3a-l were then combined
with 4-nitrophenylisocyanate to yield final compounds 4a-l. Due to production of a sticky side

product (likely a urea), purification of some final compounds proved challenging. Such reactions
were purified by silica column chromatography and then by recrystallization from acetone to
achieve high purity but with substantially reduced yields.28-31
Scheme 1. Reagents and conditions: a) pyridine, 4-AcNHPhSO2Cl; b) HCl; c) 4-NO2PhNCO
Compounds were evaluated for inhibition of Mtb NadE measuring their effect on
pyrophosphate (PPi) production using the EnzChek Pyrophosphate Assay (ThermoFisher,
Waltham, MA). Briefly, PPi formation is coupled to the production of
2-amino-6-mercapto-7-methylpurine at 360 nm. The IC50 values are shown in Table 2. The
synthesized urea-sulfonamide compounds show a 10-fold range of activity against Mtb NadE.
Parent compound 4i (5824) shows poor inhibition of Mtb NadE (IC50 = 1000
300 µM),
contrary to its more potent inhibition of B. anthracis NadE (IC50 = 6.4 µM).21 This difference in
efficacy is likely due to structural differences between the two homologs (as noted above). Our
modeling results show that the binding site of this series of compounds may involve the
intramolecular ammonia tunnel of the Mtb NadE. This tunnel is absent in B. subtilis and B.
anthracis. Thus, compound 4i likely binds to an alternate site in the Bacillus homologs compared
with Mtb NadE.

Table 2. Mtb NadE inhibition and growth inhibition of urea-sulfonamide analogs
Mtb NadE
IC50 (µM)
Mtb NadE
IC50 (µM)
Mtb MIC
(µg/mL)
7H9
Mtb MIC
(µg/mL)
GAST-Fe
Compound
R
Gln-dependent NH3-dependent
4-OH
160 20
190 10
170 20
37
100
25
25
19
37
37
37
25
50
50
4a
4b
4c
4d
4e
4f
3-CF3, 4-NO2
3-NO2
4-OMe
4-F
12.5
50
130 10
170 20
>200
>200
>200
25
4-NO2
4-CN
90
5
25
190 10
130 20
37
4g
4h
4i
3-CF3
3,4-diCl
(582420)
4-I
50
1000 300
50
370 10
37
25
50
37
4j
4k
4-Cl
520 100
H
125
5
>200
75
12.5
0.03
4l
INH
0.05
Mtb = Mycobacterium tuberculosis; IC50 = inhibitor concentration resulting in 50% enzyme inhibition; MIC =
minimum inhibitory concentration; 7H9 = rich media; GAST = minimal media; INH= isoniazid

The new analogs show improved Mtb NadE inhibition relative to the parent compound
with most IC50 values roughly 5-10-fold better than 4i. The most potent Mtb NadE inhibitor of
the series is 4f (4-NO2) with an IC50 value of 90 µM. Comparing unsubstituted analog 4l with
other analogs in the series, halogen-substituents are generally not beneficial in terms of Mtb
NadE inhibition as compounds 4e, 4j, and 4k were among the least potent of the series. The
remaining analogs, 4a-d and 4f-h have similar activities compared to unsubstituted compound 4l
and yield a bland structure-activity-relationship profile between these substituents and Mtb NadE
inhibition.
To further investigate the binding mechanism of this series of compounds, an
32
ammonia-dependent Mtb NadE inhibition assay12,
was performed on the most potent
compounds 4d, 4f, 4h and 4l. In this assay, exogenous ammonia is used as the ammonia source
for the NadE reaction, as opposed to the ammonia synthesized by the glutaminase domain of the
enzyme. As shown in Table 2, these compounds lose inhibition (IC50 >200 µM) against Mtb
NadE when exogenous ammonia is supplied, and enzymatic activity is restored. This supports
our hypothesis that these compounds bind to the intramolecular ammonia tunnel suggested from
the modeling study, as well as offering an explanation of the activity difference observed for
compound 4i against B. anthracis NadE versus Mtb NadE.
The urea-sulfonamides were also tested for the inhibition of Mtb cellular growth in both
rich media (7H9) and minimal media (GAST-Fe).33-36 The antitubercular activities of these
compounds are shown in Table 2. All compounds inhibit Mtb growth to a moderate extent with

MIC values of 19-100 µg/mL (7H9). The most potent compound is 4e (4-F) with an MIC value
of 19 µg/mL (7H9). Again, the structure-activity relationship of the series is flat, indicating this
portion of the urea-sulfonamides does not impact antitubercular activities to a great extent.
There remains an urgent need for antitubercular drugs that are highly active and work
through new modes of actions. This work builds on the novel design of three-ring
urea-sulfonamides as NadE inhibitors. The parent compound 4i, shows very different potency
against Mtb and B. anthracis NadE homologs, perhaps due to structural differences between the
two enzymes. This is supported by our preliminary modeling studies, showing that the binding
sites of these compounds may overlap with the ammonia tunnel in Mtb NadE rather than the
NaAD+ subsite in B. anthracis NadE20, as suggested earlier. This hypothesis is reinforced by the
loss of activities for the active Mtb NadE inhibitors when treated with exogenous ammonia
instead of that from glutamine. This point of difference may be used to design selective
inhibitors targeting Mtb NadE over other homologs. While most of the analogs display a
5-10-fold improvement against Mtb NadE over the parent compound, all of the compounds show
moderate activity against Mtb whole cell growth. Structural optimization of these compounds is
needed, not only to improve activity against both Mtb NadE and Mtb, but also to increase
solubility which was noticeably poor in various organic solvents and water. Such compounds
would aid in further validating NadE as an effective target for Mtb and other bacterial pathogens.
Acknowledgments

We thank Nigel Richards (Cardiff University) for helpful discussions throughout this project.
This work was generously supported by the George Washington University (GWU) Department
of Chemistry, the GWU University Facilitating Fund, the American Chemical Society (PRF to
BG), the University of Maryland College Park Department of Chemistry and Biochemistry, and
the Division of Intramural Research, NIAID, NIH.
Supplementary data
Supplementary data (experimental procedure of the synthesis, NadE inhibition assay, protein
sequence analysis, and modeling studies) associated with this article can be found, in the online
version, at xxxxx.

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28. Compound 4e. To a solution of the 3e (1 eq) in anhydrous THF (30 mL/mmol) was added
4-nitrophenylisocyanate (1 eq). The reaction mixture was stirred at room temperature overnight, concentrated.
Silica cake was prepared in acetone and chromatographic separation on silica gel (EtOAc/CH2Cl2 = 1/30 to 1/5)
gave the crude solids which were further recrystallized in acetone to give the expected compound as white solids.
Yield: 14%. 1H NMR (acetone-d6, 400 MHz), δ (ppm): 6.99-7.07 (m, 2H), 7.20-7.25 (m, 2H), 7.63-7.71 (m, 4H),
7.76-7.81 (m, 2H), 8.17-8.23 (m, 2H), 8.74 (s, 1H), 8.89 (s, 1H), 8.93 (s, 1H). 13C NMR (acetone-d6, 100 MHz), δ
(ppm): 116.53, 116.76, 119.02, 119.14, 124.57, 124.65, 125.84, 129.36, 133.97, 135.20, 135.23, 143.31, 144.52,
146.83, 152.62, 159.79, 162.19. LC/MS (ESI) m/z: 431 (M+1), 861 (2M+1).
29. Compound 4f. To a solution of the 3f (1 eq) in anhydrous THF (30 mL/mmol) was added
4-nitrophenylisocyanate (1 eq). The reaction mixture was stirred at room temperature overnight, concentrated.
Silica cake was prepared in acetone and chromatographic separation on silica gel (EtOAc/CH2Cl2 = 1/30 to 1/5)
gave the crude solids which were further recrystallized in acetone to give the expected compound as yellow solids.

Yield: 7%. 1H NMR (acetone-d6, 400 MHz), δ (ppm): 7.48-7.54 (m, 2H), 7.74-7.85 (m, 4H), 7.88-7.94 (m, 2H),
8.18-8.27 (m, 4H), 8.81 (s, 1H), 8.94 (s, 1H), 9.79 (s, 1H). 13C NMR (acetone-d6, 100 MHz), δ (ppm): 118.95, 119.27,
119.31, 125.72, 126.00, 129.40, 133.44, 143.26, 144.99, 145.25, 146.60, 150.95, 152.43. LC/MS (ESI) m/z: 458
(M+1), 915 (2M+1).
30. Compound 4h. To a solution of the 3h (1 eq) in anhydrous THF (30 mL/mmol) was added
4-nitrophenylisocyanate (1 eq). The reaction mixture was stirred at room temperature overnight, concentrated.
Silica cake was prepared in acetone and chromatographic separation on silica gel (EtOAc/CH2Cl2 = 1/30 to 1/5)
gave the crude solids which were further recrystallized in acetone to give the expected compound as light yellow
solids. Yield: 13%. 1H NMR (acetone-d6, 400 MHz), δ (ppm): 7.26-7.33 (m, 1H), 7.38-7.42 (m, 2H), 7.43-7.48 (m,
1H), 7.58-7.63 (m, 2H), 7.65-7.70 (m, 4H), 8.06-8.13 (m, 2H), 8.60 (s, 1H), 8.76 (s, 1H), 9.19 (s, 1H). 13C NMR
(acetone-d6, 100 MHz), δ (ppm): 117.33, 118.92, 119.18, 121.49, 124.49, 125.71, 129.28, 131.19, 133.59, 139.91,
143.22, 144.70, 146.62, 152.42. LC/MS (ESI) m/z: 481 (M+1).
31. Compound 4l. To a solution of the 3l (1 eq) in anhydrous THF (30 mL/mmol) was added
4-nitrophenylisocyanate (1 eq). The reaction mixture was stirred at room temperature overnight, concentrated.
Silica cake was prepared in acetone and chromatographic separation on silica gel (EtOAc/CH2Cl2 = 1/30 to 1/5)
gave the crude solids which were further recrystallized in acetone to give the expected compound as white solids.
Yield: 8%. 1H NMR (acetone-d6, 400 MHz), δ (ppm): 7.04-7.08 (m, 1H), 7.20-7.29 (m, 4H), 7.64-7.82 (m, 6H),
8.18-8.23 (m, 2H), 8.66 (s, 1H), 8.85 (s, 1H), 8.87 (s, 1H). 13C NMR (acetone-d6, 100 MHz), δ (ppm): 119.00, 119.08,
119.14, 119.22, 121.78, 125.39, 125.90, 129.41, 130.14, 134.50, 139.18, 144.49, 152.57. LC/MS (ESI) m/z: 413
(M+1), 825 (2M+1).
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Dowd, C. S. Structure−Acꢁvity Relaꢁonships of Anꢁtubercular Nitroimidazoles. 1. Structural Features Associated
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H. I.; Couch, R. D.; Dowd, C. S. Design of potential bisubstrate inhibitors against Mycobacterium tuberculosis (Mtb)
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Graphical Abstract
Design, synthesis, and evaluation of nicotinamide adenine dinucleotide (NAD+) synthetase
inhibitors as potential antitubercular agents
Xu Wang, Yong-Mo Ahn, Adam G. Lentscher, Julia S. Lister, Robert C. Brothers, Malea M.
Kneen, Barbara Gerratana, Helena I. Boshoff, Cynthia S. Dowd