Aryl-alkyl-lysines: Membrane-Active Fungicides That Act against Biofilms of Candida albicans

Article abstract of DOI:10.1021/acsinfecdis.6b00192

Mortality due to pathogenic fungi has been exacerbated by the rapid development of resistance to frontline antifungal drugs. Fungicidal compounds with novel mechanisms of action are urgently needed. Aryl-alkyl-lysines, which are membrane-active small molecules, were earlier shown to be broad-spectrum antibacterial agents with potency in vitro and in vivo. Herein, we report the antifungal properties of aryl-alkyl-lysines. After identifying the most active compound (NCK-10), we tested its activity against a panel of clinically relevant pathogenic fungi and examined NCK-10's effect against immature and mature biofilms of Candida albicans. NCK-10 was capable of inhibiting the growth of various species of fungi (including Candida spp., Cryptococcus spp., and Aspergillus fumigatus) at concentrations similar to those of antifungal drugs used clinically. It was observed that polarization and permeability of the fungal cell membrane were compromised upon addition of NCK-10, indicating its mechanism is disruption of the fungal cell membrane. In addition to interfering with the growth of planktonic fungi, NCK-10 demonstrated the ability to both inhibit biofilm formation and reduce the metabolic activity of cells in C. albicans biofilm. Additionally, our compound was capable of crossing the blood-brain barrier in an in vitro model, expanding the potential antifungal applications for NCK-10. Overall, aryl-alkyl-lysines were found to be excellent compounds that warrant further investigation as novel antifungal agents.

Full text of DOI:10.1021/acsinfecdis.6b00192

Subscriber access provided by University of Newcastle, Australia
Article
Aryl-alkyl-lysines: Membrane-active fungicides
that act against biofilms of Candida albicans
Chandradhish Ghosh, Vikas Yadav, Waleed Younis, Haroon Mohammad,
Youssef A. Hegazy, Mohamed N. Seleem, Kaustuv Sanyal, and Jayanta Haldar
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.6b00192 • Publication Date (Web): 27 Feb 2017
Downloaded from http://pubs.acs.org on March 1, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Infectious Diseases is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 35
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
Arylꢀalkylꢀlysines: Membraneꢀactive fungicides that
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
act against biofilms of Candida albicans
Chandradhish Ghosh,a Vikas Yadav,b Waleed Younis,c Haroon Mohammad,c Youssef A. Hegazy,c
Mohamed N. Seleem,c,d Kaustuv Sanyal,b and Jayanta Haldar a*
aChemical Biology and Medicinal Chemistry Laboratory, New Chemistry Unit, Jawaharlal
Nehru Centre for Advanced Scientific Research, Jakkur, Bengaluru 560064, Karnataka, India;
bMolecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru
Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India
cDepartment of Comparative Pathobiology, Purdue University, 625 Harrison Street, West
Lafayette, Indiana 47907, USA
dPurdue Institute for Inflammation, Immunology, and Infectious Disease, Purdue University,
West Lafayette, IN 47907, USA.
Corresponding author: Jayanta Haldar (Email: jayanta@jncasr.ac.in)
ABSTRACT: Mortality due to pathogenic fungi has been exacerbated by the rapid development
of resistance to frontline antifungal drugs. Fungicidal compounds with novel mechanisms of
action are urgently needed. Arylꢀalkylꢀlysines, which are membraneꢀactive small molecules,
1
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 2 of 35
1
2
3
4
5
6
7
8
9
were earlier shown to be broadꢀspectrum antibacterial agents with potency in vitro and in vivo.
Herein, we report the antifungal properties of arylꢀalkylꢀlysines. After identifying the most active
compound (NCKꢀ10), we tested its activity against a panel of clinically relevant pathogenic fungi
and examined NCKꢀ10’s effect against immature and mature biofilms of Candida albicans.
NCKꢀ10 was capable of inhibiting growth of various species of fungi (including Candida spp.,
Cryptococcus spp., and Aspergillus fumigatus) at concentrations similar to antifungal drugs used
clinically. It was observed that polarization and permeability of the fungal cell membrane was
compromised upon addition of NCKꢀ10, indicating its mechanism is disruption of the fungal cell
membrane. In addition to interfering with growth of planktonic fungi, NCKꢀ10 demonstrated the
ability to both inhibit biofilm formation and reduce the metabolic activity of cells in C. albicans
biofilm. Additionally, our compound was capable of crossing the bloodꢀbrainꢀbarrier in an in
vitro model, expanding the potential antifungal applications for NCKꢀ10. Overall, arylꢀalkylꢀ
lysines were found to be excellent compounds that warrant further investigation as novel
antifungal agents.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Key words: Antimicrobial peptides, Antifungal drugs, Fungal Biofilms, Peptidomimetics
INTRODUCTION
Fungal infections afflict millions of patients annually with more than 50% mortality associated
with invasive fungal infections.1 Despite the fact that infections caused by pathogenic fungi lead
to significant mortality and morbidity, the threat of these pathogens is often overlooked.1 Only
four classes of antifungal drugs are currently available to clinicians: polyenes, azoles,
echinocandins and 5ꢀfluorocytosine.2 While azoles inhibit the biosynthesis of ergosterols,
2
ACS Paragon Plus Environment

Page 3 of 35
ACS Infectious Diseases
1
2
polyenes bind to ergosterol, form pores, and induce leakage of intracellular ions.2ꢀ3 In contrast,
echinocandins and 5ꢀfluorocytosine exert their antifungal effect by a different method.
Echinocandins inhibit fungal cell wall biosynthesis while 5ꢀfluorocytosine inhibits both protein
synthesis and DNA synthesis in fungi.4 There are reports of resistance to each antifungal agent
mentioned above; this makes it imperative to design and develop novel antifungal agents.5
In additional to targeting fungal viability, identifying novel antifungals capable of interfering
with key fungal virulence factors (such as biofilm formation) is highly desirable. Similar to
bacteria, fungi are capable of forming stable communities within biofilms that are extremely
difficult to treat.6 Only recently have therapeutic agents against antifungal biofilms gained more
prominence in the research community.7ꢀ8 Thus design of novel antifungal agents which can act
against both planktonic and biofilm forms of pathogenic fungi is highly desirable.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Antimicrobial peptides and their mimics have gained popularity among researchers as
next generation therapeutic agents, primarily because of their broad spectrum of activity.9ꢀ10
Several antimicrobial peptides possess notable antifungal activity. This has prompted additional
research in peptidomimetic designs with antifungal properties.11ꢀ13 Although, none of the
peptidomimetic designs have progressed to preclinical studies to date, they bear much promise as
potential drugs for the treatment of pathogenic fungi.
Our research group has designed several macromolecular and small molecule membraneꢀ
active agents which are potent antimicrobial agents alone and in combination with traditional
antibiotics.14ꢀ16 Interestingly, most of these agents also possess potent antibiofilm properties
against both Gramꢀpositive and Gramꢀnegative bacteria.17ꢀ18 Herein, we have explored the
antifungal properties of one subset of bioactive molecules in our compound library, arylꢀalkylꢀ
lysines. Arylꢀalkylꢀlysines are compounds consisting of two hydrophobic groups (a lipid tail and
3
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 4 of 35
1
2
an aromatic head group) and two positive charges (contributed by LꢀLysine).19 They were earlier
shown to be potent antibacterial agents both in vitro and in vivo.17ꢀ18 Herein, we tested the
activity of compounds from the NCK series, containing naphthalene rings, and the BCK series,
containing benzene rings, against clinicallyꢀrelevant pathogenic fungi. After identification of the
most active compound (NCKꢀ10), its antifungal mechanism of action was confirmed.
Furthermore, the ability of the compound to inhibit C. albicans biofilms was also examined.
Finally, we studied if NCKꢀ10 was capable of passively permeating across the relatively
impermeable bloodꢀbrainꢀbarrier, using an in vitro model. The results garnered provide a
valuable foundation for further investigation for potential clinical applications of NCKꢀ10 as a
novel antifungal agent.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
RESULTS AND DISCUSSION
Synthesis: Arylꢀalkylꢀlysines were synthesized and characterized using a previously published
protocol.19 They were a robust ensemble of a naphthalene or a benzene moiety, a lipophilic chain
and an LꢀLysine moiety (Figure 1). Herein, we have studied the efficacy of representative
compounds bearing the naphthalene ring (NCK series) or benzene ring (BCK series). In the NCK
series, the length of the lipophilic tail was varied from hexyl to decyl group to yield compounds
NCKꢀ6, NCKꢀ8 and NCKꢀ10. In the BCK series, the lipid tail was varied from decyl to
tetradecyl moiety to obtain compounds BCKꢀ10, BCKꢀ12 and BCKꢀ14.
Antifungal activity:
The antifungal activity of the six compounds presented above were initially screened against
two fungal strains, C. albicans SC5314 and C. neoformans H99, in YPD media. The MIC
(minimum inhibitory concentration or the concentration at which the growth of the fungal cells
are inhibited) and MFC (minimum fungicidal concentration or the concentration of the
4
ACS Paragon Plus Environment

Page 5 of 35
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
compound at which fungal cells are killed) of the compounds is presented in Table 1. NCKꢀ6
lacked antifungal activity against C. albicans SC5314. The MIC for NCKꢀ8 was 25 µg mLꢀ1 and
correlated to the MFC, indicating this compound is fungicidal at this concentration. From the
NCK series, NCKꢀ10, was the most potent antifungal compound identified, as it inhibited growth
of C. albicans SC5314 at a concentration of 12.5 µg mLꢀ1. In the BCKꢀseries, BCKꢀ12 showed
potent activity inhibiting fungal growth at 25 µg mLꢀ1 and demonstrating a fungicidal effect at 25
µg mLꢀ1. Although the decyl analogue BCKꢀ10 was slightly active against C. albicans (MIC of
25 µg mLꢀ1), BCKꢀ14 was inactive even at a concentration of 50 µg mLꢀ1. Against C.
neoformans H99, however, all the compounds (except for NCKꢀ6 and BCKꢀ14) inhibited growth
at 3.1 µg mLꢀ1. From this screen, NCKꢀ10 emerged as the most active compound against both C.
albicans and C. neoformans. Thus, we moved to further explore the spectrum of antifungal
activity of NCKꢀ10.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The susceptibility of cationic antimicrobial agents often vary in different media.20 We have
reported similar observations against bacteria previously.21ꢀ22 In order to test if YPD media had
an adverse effect with respect to susceptibility, we determined the MIC and MFC of NCKꢀ10 in
RPMI medium as well. Interestingly, a significant decrease in the MIC value was observed. The
MIC of the compound against C. albicans SC5314 in RPMI media was found to be 1.56 µg mLꢀ
1. Thus, we chose to carry out further susceptibility testing using RPMI medium. NCKꢀ10 was
subsequently tested against additional clinical fungal species including C. albicans, C.
parapbsilosis, C. glabrata, C. tropicalis, C. gattii, C. neoformans and A. fumigatus (Table 2).
Susceptibility of nine strains of C. albicans to NCKꢀ10 was examined. Out of these nine strains,
two strains (NRꢀ29351 and NRꢀ29365) were susceptible to fluconazole (MIC of 0.5 µg mLꢀ1).
Against NRꢀ29351, NCKꢀ10 was active at 1 µg mLꢀ1 while amphotericin B was active at 0.5 µg
5
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 6 of 35
1
2
mLꢀ1. Against NRꢀ29365, however, NCKꢀ10 (MIC 0.5 µg mLꢀ1) was fourꢀfold more active than
amphotericin B (MIC 2 µg mLꢀ1). Against the remaining seven strains of C. albicans,
fluconazole was not active up to a concentration of 64 µg mLꢀ1. Against C. albicans NRꢀ29448,
NCKꢀ10 matched the potency of amphotericin B, inhibiting fungal growth at a concentration of
0.5 µg mLꢀ1. Against C. albicans ATCC MYA A573, NCKꢀ10 (MIC 0.5 µg mLꢀ1) was twoꢀfold
more active than amphotericin B. When examined against C. albicans NRꢀ29438, NRꢀ29366,
NRꢀ29367, ATCC 26790 and ATCC 64124, amphotericin B inhibited growth at 1 µg mLꢀ1 which
was similar to the MIC for NCKꢀ10 (2 µg mLꢀ1).
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Against C. parapsilosis ATCC 22019, NCKꢀ10 had MIC and MFC values of 0.5 µg mLꢀ1 and
1 µg mLꢀ1 respectively, which is comparable to the control antifungals amphotericin B (MIC of
0.5 µg mLꢀ1 and MFC of 2 µg mLꢀ1) and superior to fluconazole (MIC and MFC of 2 µg mLꢀ1).
Against two strains of C. glabrata (ATCC MYAꢀ2950 and ATCC 66032), which were resistant
to fluconazole (MFC >64 µg mLꢀ1), NCKꢀ10 was active at 1 µg mLꢀ1 (MIC and MFC). These
strains were susceptible to amphotericin B (MIC 0.5ꢀ1 µg mLꢀ1). We tested the activity of NCKꢀ
10 against two species of C. tropicalis ATCC 1369 and ATCC 13803. Against both strains,
amphotericin was active at 0.5 µg mLꢀ1. NCKꢀ10 was also active at 0.5 µg mLꢀ1 against ATCC
1369 while fluconazole was active at 2 µg mLꢀ1. Against ATCC 13803, fluconazole was active at
0.5 µg mLꢀ1 while NCKꢀ10 was active at 1 µg mLꢀ1. Overall, the potency of NCKꢀ10 against
Candida species was similar to, and against several strains better than, the clinical antifungals
amphotericin B and fluconazole.
Against two strains of C. gattii (NRꢀ43208 and NRꢀ43209), NCKꢀ10 and amphotericin B
exhibited identical MIC values of 1 µg mLꢀ1, whereas the MIC of fluconazole was 2 µg mLꢀ1 and
8 µg mLꢀ1 respectively. Against four strains of C. neoformans (NRꢀ41292, NRꢀ41294, NRꢀ41295
6
ACS Paragon Plus Environment

Page 7 of 35
ACS Infectious Diseases
1
2
and NRꢀ41296), the MIC of fluconazole varied from 4 µg mLꢀ1 to 16 µg mLꢀ1. The antifungal
activity of NCKꢀ10 against these same strains was superior (MIC ranged from 1 µg mLꢀ1 to 2 µg
mLꢀ1) and was comparable to the activity of amphotericin B (MIC ranged from 0.5 µg mLꢀ1 to 1
µg mLꢀ1).
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Similar to fungal pathogens like C. neoformans, Aspergillus fumigatus severely affects
immunocompromised individuals including patients with leukemia, cystic fibrosis, human
immunodeficiency virus, and patients undergoing organ and stem cell transplants; mortality rates
associated with this pathogen are as high as 90%.23 We tested NCKꢀ10 against five strains of A.
fumigatus (NR 35301, NR 35302, NR 35303, NR 35304 and NR 35308) which were all resistant
to fluconazole (MIC >64) but susceptible to amphotericin B (MIC 0.5 µg mLꢀ1 or less). Against
all strains of A. fumigatus that were tested, NCKꢀ10 was fungicidal at 4 µg mLꢀ1.
Fungicidal time-kill kinetics: Per the MFC data, NCKꢀ10 appears to be a fungicidal agent
against all fungal species tested. We moved to confirm this observation via a standard timeꢀkill
assay. The growth of C. albicans SC5314 in the presence of NCKꢀ10 (at concentrations
equivalent to the MIC and 4×MIC) was observed over a fiveꢀhour period. As depicted in Figure
2, NCKꢀ10 was effective in diminishing the fungal count within the first hour. At the end of
three hours >90% was observed. Although, a few colonies were observed thereafter, the ability
of the compound to lyse fungal cells was evident.
Mechanism of action: As mentioned earlier, most antibiotics require very high concentrations
to have sufficient activity against stationary phase bacteria.24 NCKꢀ10 when treated against E.
coli cells in stationary phase, showed complete killing even at its MIC. While in the control, the
bacterial cell count was maintained at 5 log CFU, no colonies were observed in NCKꢀ10 treated
cultures (the limit of detection in this experiment is 50 CFU mLꢀ1.
7
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 8 of 35
1
2
3
4
5
6
7
8
9
We previously confirmed NCKꢀ10 exerted its antibacterial effect by infiltrating the cell
membrane.17ꢀ18 Thus, we postulated that NCKꢀ10 would behave in a similar manner against
fungi. To confirm this, the compound’s ability to depolarize and permeabilize the fungal cell
membrane of C. albicans (SC5314) was examined (Figure 3). The ability of NCKꢀ10 to perturb
the polarization across fungal membranes was studied using the membrane potential sensitive
probe, DiSC3(5). Although the fluorescence of the dye is initially quenched upon being taken up
by fungal cells, the fluorescence intensity of the dye is enhanced upon perturbation of the
membrane potential. NCKꢀ10 at concentrations equivalent to its MIC and 2 × MIC was able to
depolarize the fungal membrane within one minute (Figure 3A), in a doseꢀdependent manner.
We then checked if the compound was able to compromise the membrane to permit influx of
external substances. The external agent used was propidium iodide (PI), which cannot enter the
cell unless the cell membrane is compromised. Upon binding to nucleic acids, there is an
enhancement in the fluorescence signal emitted. Upon addition of the compound, PI found rapid
entry inside the fungal cell as presented in Figure 3B. These results support our hypothesis that
NCKꢀ10 exerts its antifungal effect by rapidly permeabilizing the fungal cell membrane.
Microscopic observation of cell viability
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In order to further confirm the proposed antifungal mechanism of action, NCKꢀ10’s ability to
compromise the fungal cell membrane was observed microscopically. In this experiment, two
dyes SYTOꢀ9 and PI were used to differentiate between live and dead/membraneꢀcompromised
cells. SYTOꢀ9 gains entry both in intact and membraneꢀcompromised cells. However, as
mentioned earlier, PI can only enter membraneꢀcompromised cells. As can be seen from Figure
4, in the untreated control, only uptake of SYTOꢀ9 by C. albicans SC5314 was observed,
indicating that the cell membrane was not compromised. However, upon treatment of C. albicans
8
ACS Paragon Plus Environment

Page 9 of 35
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
SC5314 cells with NCKꢀ10 (at its MIC)), PI entered the cells easily. In the merged image, one
can see the predominance of the red dye (PI) in comparison to the untreated control, further
confirming the membraneꢀdisrupting ability of NCKꢀ10.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Anti-biofilm properties
The ability of the compound to both prevent and disrupt biofilms of C. albicans SC5314 was
tested. To examine inhibition of biofilm formation, we incubated NCKꢀ10 at different subꢀMIC
concentrations (ranging from MIC to MIC/32) and allowed the fungal biofilms to grow for three
days. Subsequently, the metabolic activity of biofilm that was formed was quantified using the
MTT assay. As presented in Figure 5A, with respect to the control, the compound was able to
inhibit formation of biofilms in a concentrationꢀdependent manner, At MIC/32, more than 30%
inhibition of fungal biofilm formation was observed. At MIC/4 the compound produced an 80%
inhibition of biofilm formation, relative to the untreated control. The effect was most pronounced
at MIC/2 and MIC/4 concentrations.
We next investigated the efficacy of the compound to disrupt preꢀformed fungal biofilm. C.
albicans SC5314 biofilms were grown for 48 hours before subjecting them to compound
treatment. The concentration of the compound tested ranged from MIC/4 to 4 × MIC. The
metabolic activity of the biofilm was significantly diminished by 25% even at MIC/2. No
antibiofilm activity was observed for NCKꢀ10 at MIC/4 (Figure 5B). At concentrations equal to
the MIC and beyond, more than 80% reduction in the metabolic activity of cells in the biofilm
was observed, relative to the untreated control.
Cytotoxicity
In order to justify the selectivity of the compounds towards fungal cells in comparison to
mammalian cells, we conducted the cytotoxicity of the most active compound, NCKꢀ10 against
9
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 10 of 35
1
2
human colorectal cells (HRTꢀ18). Till 20 µg mLꢀ1 the compound was not at all toxic to the cells
(Figure 6). This represents a 40ꢀfold difference between the MIC values obtained against some
Candida albicans strains for the compound under same conditions. The assay showed that the
effective concentration at which 50 % of the cells were killed by the compound was 42 µg mLꢀ1.
This experiment proved that the compound was selectively toxic toward fungal cells over
mammalian cells.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Parallel artificial membrane permeability assay (PAMPA) for ability to cross the blood
brain barrier (BBB)
Certain fungal infections, particularly Cryptococcal meningitis and Rhinocerebral
mucormycosis, necessitate that a viable therapeutic agent be capable of crossing the BBB in
order to reach the target site of infection. Thus we evaluated the ability of compounds NCKꢀ10
to penetrate the BBB utilizing PAMPAꢀBBB (Table 3). Although the value obtained for NCKꢀ10
was not as high as that of the highly permeable drug verapamil, it was found to be superior to the
low permeability control drug, atenolol, indicating NCKꢀ10 is capable of passively traversing
across the barrier imposed by the blood brain barrier. Traditionally, compounds possessing a
mean permeability coefficient less than 2 × 10ꢀ6 cm secꢀ1 will experience difficulty in effectively
crossing the BBB. As NCKꢀ10, possesses a mean permeability coefficient (Table 3) of 2.87 × 10ꢀ
cm secꢀ1, this indicates that NCKꢀ10 would be a good candidate for investigation of treatment
6
of fungal infections impacting the brain (namely meningitis).
DISCUSSION
Pathogenic fungi are notorious for causing both superficial and lifeꢀthreatening infections both
in healthy and immunocompromised individuals.25 More than 300 million individuals worldwide
are afflicted with a serious fungal infection. Although infections caused by bacteria, viruses and
10
ACS Paragon Plus Environment

Page 11 of 35
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
parasites are often highlighted, infections caused by pathogenic fungi have largely remained out
of limelight. The emergence of resistance to frontꢀline antifungal drugs (including fluconazole)
has further exacerbated the issue. This necessitates the identification and development of new
antifungal agents to address this significant public health challenge. Membraneꢀactive agents
have shown much promise as broadꢀspectrum antimicrobial agents.26 However, few synthetic
membraneꢀactive agents have been investigated against pathogenic fungi. Arylꢀalkylꢀlysines
have been shown to be safe and effective antibacterial compounds with potent activity in murine
models of infection.17ꢀ18 Given their membraneꢀactive nature we surmised that arylꢀalkyꢀlysines
would be potent antifungal agents capable of treating fungal infections as well.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Initially we screened six compounds from our library against the most common fungi
encountered in clinical infections, C. albicans and C. neoformans. Though most of the
compounds possessed antifungal activity, NCKꢀ10 was found to be the most active compound
(similar to previous studies involving pathogenic bacteria). We moved to conduct a more robust
analysis by examining the antifungal activity of NCKꢀ10 against a panel of fungal clinical
isolates that encompasses most of the pathogenic fungi encountered in infections in hospitals.
Candida parapsilosis has been gaining notoriety as an invasive fungus responsible for the most
infections caused by Candida spp. after C. albicans.27 Furthermore, excessive use of
immunosuppressive agents and broadꢀspectrum antimycotic agents has led to emergence of
pathogenic strains of C. glabrata, especially for patients suffering from HIV and diabetes.28 A
major problem with this microorganism is that some of the strains are inherently resistant to
fluconazole therapy. Candida tropicalis is another major problem in India, wherein it is the most
dominant contributor to candidiasis caused by Candidaꢀnonꢀalbicans (CAN) species.
Cryptococcosis and cryptococcal meningitis are potentially fatal diseases caused by
11
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 12 of 35
1
2
3
4
5
6
7
8
9
Cryptococcus spp. Although Cryptococcus neoformans mostly affects immunocompromised
individuals (particularly patients infected with HIV), Cryptococcus gattii has been reported to
cause illness in immunocompetent individuals.29 Drugs against such pathogens are urgently
needed, particularly in SubꢀSaharan Africa where the largest population of AIDS patients
resides.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Notably, several strains were resistant to antifungal drugs including fluconazole. It is evident
from MIC and MFC values disclosed in Tables 2 that the efficacy of the compound was
comparable to that of the marketed drugs amphotericin B and fluconazole against C. albicans, C.
parapsilosis, C. tropicalis, C. neoformans, C. gatti, and A. fumigatus. Against certain isolates,
NCKꢀ10 was more active than both fluconazole and amphotericin B.
Previously we demonstrated in bacteria that NCKꢀ10 exerts its antibacterial effect by
targeting the cell membrane. In the present study, we confirmed that the antifungal mechanism of
action of NCKꢀ10 is similar, as the fluorescence intensity of the membrane impermeable dye PI
significantly increased in the presence of C. albicans cells treated with NCKꢀ10, within one
minute. This observation was confirmed visually using fluorescence microscopy as a significant
increase of PI was observed inside C. albicans cells exposed to NCKꢀ10, in contrast to untreated
cells. Our preliminary investigation into the antifungal mechanism of action of NCKꢀ10,
however does not preclude the possibility that NCKꢀ10 also exerts its effect by targeting other
cellular processes. Indeed, a chemical proteomics study with the arylꢀalkylꢀlysine compounds
may indeed shed light into their ability to bind to important fungal proteins. A detailed
investigation in relation to the mechanism of action of these compounds against fungi is
currently underway. It is also clear though, that the rapid fungicidal action of the compounds can
be attributed to its membraneꢀactive nature. NCKꢀ10 was shown to be not toxic to mice at
12
ACS Paragon Plus Environment

Page 13 of 35
ACS Infectious Diseases
1
2
3
4
therapeutically relevant concentrations and herein we have shown that there is substantial
5
6
17ꢀ19
selectivity against fungal cells over HRT cells (Figure 6).
This selectivity also emphasizes
7
8
9
on the potential of the compound as a template for design of future drugs.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
PAMPA is a valuable tool in earlyꢀstage drug discovery to evaluate the ability of compounds
to passively permeate across biological membranes (such as the BBB). The PAMPAꢀBBB model
mimics the rigid, hydrophobic nature of the BBB membrane and has been shown to correlate
30ꢀ32
well with the more laborious in-situ brain perfusion assay.
One of the most interesting
characteristics discovered in this study is NCKꢀ10 appears to be capable of passively permeating
across the BBB. Though further in vivo studies need to be conducted, this observation potentially
opens the door to use NCKꢀ10 for treating both fungal and bacterial infections impacting the
brain (such as meningitis). One caveat of the PAMPAꢀBBB method is it does not account for
potential interaction of compounds with membrane proteins that help transport drugs across the
BBB. Thus, further investigation to evaluate the impact of membrane transporters to enhance
permeation of NCKꢀ10 across the BBB needs to be conducted.
In addition to the challenge posed by freeꢀfloating, planktonic fungal cells, fungal
biofilms pose a significant additional concern. These biofilms are capable of forming on medical
devices (such as indwelling catheters) leading to recurring infections that are challenging to
treat.33 Understandably, several recent studies have focused on identifying agents that are
effective at attacking biofilms of pathogenic fungi.7ꢀ8, 34ꢀ35 The present study revealed that our
compound, NCKꢀ10, was capable of both inhibiting biofilm formation (at subinhibitory
concentrations) and reducing the metabolic activity of fungal cells encased in a biofilm.
Although the ability of NCKꢀ10 to inhibit and reduce the metabolic activity of preꢀformed
biofilms was investigated against C. albicans only, the antibiofilm activity of NCKꢀ10 has
13
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 14 of 35
1
2
3
4
5
6
7
8
9
potential to be extended to other pathogenic fungi as well. This is indeed an important
contribution to the field, as it is imperative to have drugs which can act against both planktonic
cells and biofilms of pathogenic fungi.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CONCLUSION
Overall, present study demonstrates the promise of arylꢀalkylꢀlysines, specifically NCKꢀ10, as
prospective antifungal agents. NCKꢀ10 possesses excellent efficacy in vitro against various
species of clinicallyꢀrelevant fungi and is also capable of interfering with biofilms formed by C.
albicans. The compound exerts its antifungal effect by targeting the cell membrane of fungi,
inducing rapid lysis. The next step towards validating the therapeutic potential of these
compounds as antifungal agents is testing their efficacy in suitable animal models of infection.
Given fungi are responsible for various infections of the body, it will be worthwhile to
investigate whether NCKꢀ10 is able to treat invasive fungal infections (e.g. candidiasis), brain
infections (caused by Cryptococcal meningitis), skin infections (onchomycosis) and pulmonary
infections. At present, NCKꢀ10 represents a good lead for design of a new generation of
antifungal agents.
MATERIALS AND METHODS
Materials and Media:
Yeast extract peptone dextrose (YPD) was obtained from HIMEDIA, India. RPMI purchased
from GIBCO, Amphotericin B, fluconazole, atenolol, verapamil, HEPES, DiSC3 (5), MTT and
Propidium iodide (PI) were purchased from SigmaꢀAldrich (St. Louis, MO). SYTO 9 dye was
purchased from Invitrogen. For optical density (OD) and fluorescence measurement, Tecan
Infinite Pro series M200 Microplate Reader was employed. An Olympus microscope (Model
BX51) and Olympus DP71 camera were used for the fungal imaging.
14
ACS Paragon Plus Environment

Page 15 of 35
ACS Infectious Diseases
1
2
3
4
Synthesis
5
6
7
8
The synthesis of the compounds was performed using a previously published protocol.19
Briefly, in the first step, aromatic aldehydes (1 eq) and alkyl amines (1 eq) were reacted in a 1:1
mixture of dry chloroform and methanol for 6 h. Following this, reductive amination was
achieved by adding Sodium borohydride (1.8 eq) to it in ice cold condition. Upon completion of
the reaction the solvents were evaporated under reduced pressure (not to dryness) and diluted
with diethyl ether. Subsequently, 2N NaOH was added to the mixture and stirred for 15 minutes.
The organic layer was separated, washed with water, brine and dried over MgSO4. To this 4N
HCl was added to obtain precipitates was observed. The solvent was completely removed and
the precipitate was dissolved in minimum volume of ethyl acetate (a few drops of methanol was
added to dissolve completely). To this hexane was added to obtain pure crystals of the target
compound.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In the second step, amide coupling reaction was performed using HBTU. Briefly, to Bocꢀ
Lys(Boc)ꢀOH (1.2 eq) in 5:2 DMF/CHCl3, N,N-Diisopropylethylamine (DIPEA, 2.5 eq) and
HBTU (1 eq) was added at 0°C. Subsequently the secondary amines (obtained from the first
step) were added (1 eq). The mixture was stirred at 0°C for 30 minutes and subsequently at RT
for 24 h typically. Upon completion of the reaction organic solvents were evaporated under
reduced pressure and the contents were dissolved in ethyl acetate. This was then washed with 0.5
M KHSO4, H2O (thrice) and brine. After passage through anhydrous Na2SO4, the organic layer
was evaporated under reduced pressure and the residue was purified using column
chromatography (only CHCl3) to obtain the product.
In the final step compounds obtained in the second step were dissolved in DCM and were
deprotected by stirring with CF3COOH (50% by volume) at RT. All the volatile components
15
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 16 of 35
1
2
3
4
5
6
7
8
9
were removed, and the product is purified by reverse phase HPLC using 0.1% Trifluoroacetic
acid (TFA) in water/acetonitrile (0ꢀ100%) as mobile phase to more than 95% purity. C18 column
(10mm diameter, 250 mm length) and UV detector (at 270 nm wavelength) were used.
Subequently, the compounds were dried and characterized by 1H NMR, 13C NMR, IR and mass
spectrometry.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Initial fungal susceptibility assay
The antifungal activity of the compounds was initially tested against Candida albicans SC5314
and Cryptococcus neoformans H99 using a previously described 96ꢀwell plate assay, with few
modifications.16 Briefly, the fungal strains were grown overnight in 5 mL YPD (1% yeast
extract, 2% peptone and 2% dextrose). Fungal growth was measured via a spectrophotometer
(OD600) and cells were diluted in fresh media (either in YPD or RPMI) to get the required
concentration of 1.3 × 105 cells mLꢀ1. An aliquot (150 µL) of the culture dilution (equivalent to 2
× 104 cells) was added to wells containing 50 µL of water or the compounds, at different
concentrations. The plates were incubated at 30 °C for 20 hours with shaking (180 rpm) to allow
sufficient growth of fungal cells. The growth of the fungi was measured (OD600) using media
alone (YPD/RPMI) as a blank. Simultaneously, 3 µL of the culture from each well was taken and
spotted onto a YPD agar plate to determine the minimum fungicidal concentration (MFC). The
concentration where the optical density of the test well was close to the value of the blank well
value was categorized as the minimum inhibitory concentration (MIC).
Antifungal activity against clinical isolates
The minimum inhibitory concentration (MIC) of the most potent compound, NCKꢀ10, was
determined against several clinical strains of Candida species, Cryptococcus species and one
16
ACS Paragon Plus Environment

Page 17 of 35
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
strain of Aspergillus fumigatus. C. albicans, Cryptococcus spp., A. fumigatus (tested from 64 ꢁg
mLꢀ1 down to 0.5 ꢁg mLꢀ1) were transferred to a 96ꢀwell plate and incubated at 37 ºC for 48
hours (or 72 hours for C. neoformans) .The MICs reported represent the lowest concentration of
each compound necessary to inhibit fungal growth, by visual inspection. For determination of the
minimum fungicidal concentration, aliquots (5 ꢁL) were transferred from wells with no growth
onto YPD agar plates. Plates were incubated at 37 ºC for 48 hours before MFC results were
recorded. Amphotericin B and fluconazole were used as control antifungals.
Kinetics study
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In order to confirm if NCKꢀ10 was fungistatic or fungicidal, a timeꢀkill experiment was
conducted. Fungal cells were grown overnight and added to a 96ꢀwell plate as described above.
Starting at the initial time point, a small aliquot (3 µL) of cells was taken and spotted onto a YPD
plate every hour (for five hours). The plates were then incubated at 30 °C for 24 hours to score
for viable cells.
Mechanism of action
Depolarization of fungal cell membrane
Briefly, 107 CFU mlꢀ1 of C. albicans SC5314 was washed and resuspended in 5 mM glucose, 5
mM HEPES buffer and 100 mM KCl solution in a 1:1:1 ratio. To this solution DiSC3 (5) dye
(obtained from SigmaꢀAldrich) was added to a final concentration of 2 µM. The fungal
suspension containing the dye (200 µL) was preincubated for 20 min in a black 96ꢀwell plate
with a transparent bottom. The fluorescence of the fungal suspension was measured (excitation
wavelength: 622 nm; emission wavelength: 670 nm) and allowed to stabilize at room
temperature before the addition of 2 µL of NCKꢀ10 (final concentration was equivalent to the
17
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 18 of 35
1
2
MIC (12.5 µg mLꢀ1) or 2 × MIC (25 µg mLꢀ1)). After addition, the fluorescence intensity was
measured every minute for 20 minutes.
3
4
5
6
7
8
9
Permeabilization of fungal cell membrane
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Propidium iodide (PI) dye (15 µM, final concentration) (obtained from SigmaꢀAldrich) was
added to a fungal suspension containing C. albicans SC5314 (~107 cells mLꢀ1) in 5 mM HEPES
and 5 mM glucose (pH 7.4). The suspension containing the dye (200 µL) was then added to the
well of a 96ꢀwell, clearꢀbottom black plate. After four minutes, NCKꢀ10 (2 µL) was added to the
solution to a final concentration equivalent to the MIC (12.5 µg mLꢀ1) or 2 × MIC (25 µg mLꢀ1).
The fluorescence intensity was measured at excitation wavelength of 535 nm (slit width: 10 nm)
and emission wavelength of 617 nm (slit width: 5 nm). The uptake of PI was detected by the
increase in fluorescence for 10 minutes; this correlates to permeabilization of the inner cell
membrane.
Microscopy assay for viability
C. albicans SC5314 was grown in a 96ꢀwell plate for six hours in the presence or absence of
NCKꢀ10 (12.5 µg mLꢀ1). The cells were harvested, washed once with sterile water, and
resuspended in 50 µL of sterile water. The cell suspension was incubated with propidium iodide
(PI, 15 µM final concentration) and SYTO9 (2 µM final concentration) at room temperature for
30 minutes. The cells were then observed under a fluorescence microscope. A brightꢀfield filter
was used for differential interference conference (DIC) images whereas green and red emission
filters were used to capture SYTO 9 and PI fluorescence, respectively. The images were captured
using an Olympus microscope (Model BX51) and an Olympus DP71 camera. The images were
further processed using Image ProꢀPlus software and ImageJ.
Inhibition of C. albicans biofilm formation
18
ACS Paragon Plus Environment

Page 19 of 35
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
Candida albicans SC5314 cells were grown overnight in YPD, pelleted and washed with 1 ×
phosphate buffered saline (PBS). Washed cells were resuspended in RPMI media to reach a final
concentration of 6.7 × 105 cells mLꢀ1. Compound NCKꢀ10 (50 µL; corresponding to its MIC
value of 12.5 µg mLꢀ1 and further dilutions) and 150 µL of cell suspension were mixed together
and added into wells of a 96ꢀwell plate. Wells lacking either compound or cell suspension were
utilized as positive and negative controls. The plates were incubated at 37 °C for 72 hours in
stationary condition. Inhibition of biofilm was then quantified using the MTT assay, as follows.
The wells were washed twice with 1 × PBS. MTT solution (100 µL of 1 mg mLꢀ1 in 1× PBS,
main stock concentration 5 mg mLꢀ1) was subsequently added to wells and incubated at 37 °C for
four hours. The MTT solution was discarded and wells were washed once with 1 × PBS. DMSO
(100 µL) was added into wells and further incubated at 37 °C in the dark for an hour. The
solution was then transferred to a fresh plate and the optical density was measured at 590 nm to
quantify the extent of biofilm inhibition compared to untreated wells.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Disruption of pre-formed C. albicans biofilms
Candida albicans SC5314 cells were grown overnight in YPD, pelleted and washed with 1 ×
PBS. Washed cells were resuspended in RPMI medium to a final concentration of 1 × 106 cells
mLꢀ1. An aliquot of cell suspension (100 µL) was added into wells and incubated at 37 °C for 24
hours. The nonꢀadhered cells were removed from the wells and 50 µL of fresh RPMI medium
along with 50 µL of varying concentrations of the compound (MIC equals to 12.5 µg mLꢀ1) were
added into the cells. As a control, 100 µL of RPMI media alone was added. These plates were
further incubated at 37 °C for 48 hours. Inhibition or disruption of biofilm was then quantified
using the MTT assay, as described above.
19
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 20 of 35
1
2
3
4
5
6
7
8
9
Compound NCKꢀ10 were assayed (at different concentrations) against a human colorectal (HRTꢀ
18) cell line to determine the potential toxic effect to mammalian cells in vitro. Cells were
cultured in RPMIꢀ1640 medium supplemented with 10% fetal horse serum at 37 °C with CO2
(5%). Control cells received DMSO alone at a concentration equal to that in drugꢀtreated cell
samples. The cells were incubated with the compound (in triplicate) in a 96ꢀwell plate at 37 ºC
with CO2 (5%) for two hours. The assay reagent MTS 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(3ꢀ
carboxymethoxyphenyl)ꢀ2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀtetrazolium) (Promega, Madison, WI, USA) was
subsequently added and the plate was incubated for four hours. Absorbance readings (at OD490)
were taken using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA, USA). The
quantity of viable cells after treatment with the compounds was expressed as a percentage of the
viability of DMSOꢀtreated control cells (average of triplicate wells ± standard deviation). The
toxicity data was analyzed via a twoꢀway ANOVA, with post hoc Dunnet’s multiple
comparisons test (P < 0.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Parallel artificial membrane permeability assay (PAMPA) for examining ability to cross
the blood brain barrier (BBB)
NCKꢀ10 and control drugs (atenolol and verapamil) were dissolved in transport buffer (pH 7.4)
to a final concentration of 10 µM. The filter membrane was coated with 4 µL of a 20 mg mLꢀ1
porcine brain lipid in dodecane. An aliquot (300 µL) of the compound solution was added to the
donor well. The acceptor well was filled with 200 µL of transport buffer (Dulbecco's Phosphate
Buffered Saline). The acceptor filter plate was carefully placed on to the donor plate and was left
undisturbed for 18 hours. Samples of the donor and acceptor wells were analyzed, in
quadruplicate, by LCꢀMS/MS and the effective permeability (Pe) was calculated as follows:
20
ACS Paragon Plus Environment

Page 21 of 35
ACS Infectious Diseases
1
2
3
4
5
6
7
8
ꢆꢇꢆꢈ
[ꢍꢎꢏꢂ]ꢈ
[ꢍꢎꢏꢂ]ꢐ
ꢀꢁꢂ ꢃ = ꢀꢁꢂ ꢅ−
ꢀꢋ ꢌ1 −
ꢑ}
ꢄ
(ꢆꢇ + ꢆ )ꢉꢊ
ꢈ
9
Where Pe is the permeability, VD and VA are the volumes of the donor and acceptor
compartments, A is the area of the membrane, t is the incubation time, and A and E subscripts on
the drug concentration terms refer to the acceptor and equilibrium concentrations, respectively.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
AUTHOR INFORMATION
Corresponding Author
*Phone: (+91) 80ꢀ2208ꢀ2565. Fax: (+91) 80ꢀ2208ꢀ2627. Eꢀmail: jayanta@jncasr.ac.in
Notes
The authors declare no competing financial interest. JNCASR has filed a patent application
based on the work described in the manuscript.
ACKNOWLEDGMENT
We thank Prof. C.N.R. Rao (JNCASR) for his constant support and encouragement.
REFERENCES
1)
Hidden killers: Human fungal infections. Sci Transl Med 2012, 4 (165), 165rv13.
2) Ghannoum, M. A.; Rice, L. B., Antifungal agents: Mode of action, mechanisms of
Brown, G. D.; Denning, D. W.; Gow, N. A. R.; Levitz, S. M.; Netea, M. G.; White, T. C.,
resistance, and correlation of these mechanisms with bacterial resistance. Clin. Microbiol. Rev.
1999, 12 (4), 501ꢀ17.
3)
agents. Clin. Microbiol. Rev. 1999, 12 (1), 40ꢀ79.
4) Denning, D. W., Echinocandins: a new class of antifungal. J. Antimicrob. Chemother.
2002, 49 (6), 889ꢀ891.
Sheehan, D. J.; Hitchcock, C. A.; Sibley, C. M., Current and emerging azole antifungal
21
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 22 of 35
1
2
3
4
5
6
7
8
9
5)
Kanafani, Z. A.; Perfect, J. R., Resistance to antifungal agents: Mechanisms and clinical
impact. Clin. Infect. Dis. 2008, 46 (1), 120ꢀ128.
6) Ramage, G.; Rajendran, R.; Sherry, L.; Williams, C., Fungal biofilm resistance. Int. J.
Microbiol. 2012, 2012, 528521.
7) Liu, R. H.; Chen, X. Y.; Falk, S. P.; Masters, K. S.; Weisblum, B.; Gellman, S. H.,
Nylonꢀ3 polymers active against drugꢀresistant Candida albicans biofilms. J. Am. Chem. Soc.
2015, 137 (6), 2183ꢀ2186.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
8)
Karlsson, A. J.; Pomerantz, W. C.; Neilsen, K. J.; Gellman, S. H.; Palecek, S. P., Effect
of sequence and structural properties on 14ꢀhelical betaꢀpeptide activity against Candida
albicans planktonic cells and biofilms. ACS Chem Biol 2009, 4 (7), 567ꢀ579.
9)
infective therapeutic strategies. Nat. Biotechnol. 2006, 24 (12), 1551ꢀ1557.
10) Zasloff, M., Antimicrobial peptides of multicellular organisms. Nature 2002, 415 (6870),
389ꢀ395.
Hancock, R. E. W.; Sahl, H. G., Antimicrobial and hostꢀdefense peptides as new antiꢀ
11)
De Lucca, A. J.; Walsh, T. J., Antifungal peptides: novel therapeutic compounds against
emerging pathogens. Antimicrob. Agents Chemother. 1999, 43 (1), 1ꢀ11.
12)
peptidomimetics with an arginineꢀ[betaꢀ(2,5,7ꢀtriꢀtertꢀbutylindolꢀ3ꢀyl)alanine]ꢀarginine motif
against Saccharomyces cerevisiae and Zygosaccharomyces bailii. FEMS Yeast Res 2015, 15 (3),
fov011.
Larsen, C. E.; Larsen, C. J.; Franzyk, H.; Regenberg, B., Antifungal properties of
13)
Bojsen, R.; Torbensen, R.; Larsen, C. E.; Folkesson, A.; Regenberg, B., The synthetic
amphipathic peptidomimetic LTX109 is a potent fungicide that disturbs plasma membrane
integrity in a sphingolipid dependent manner. PLoS One 2013, 8 (7), e69483.
14)
Polymers with tunable sideꢀchain amphiphilicity as nonꢀhemolytic antibacterial agents. Chem.
Commun. 2013, 49 (82), 9389ꢀ9391.
15)
Haldar, J., Selective and broad spectrum amphiphilic small molecules to combat bacterial
resistance and eradicate biofilms. Chem. Commun. 2015, 51 (71), 13670ꢀ13673.
Uppu, D. S.; Akkapeddi, P.; Manjunath, G. B.; Yarlagadda, V.; Hoque, J.; Haldar, J.,
Hoque, J.; Konai, M. M.; Samaddar, S.; Gonuguntala, S.; Manjunath, G. B.; Ghosh, C.;
16)
Hoque, J.; Akkapeddi, P.; Yadav, V.; Manjunath, G. B.; Uppu, D. S.; Konai, M. M.;
Yarlagadda, V.; Sanyal, K.; Haldar, J., Broad spectrum antibacterial and antifungal polymeric
paint materials: synthesis, structureꢀactivity relationship, and membraneꢀactive mode of action.
ACS Appl Mater Interfaces 2015, 7 (3), 1804ꢀ1815.
17)
Ghosh, C.; Manjunath, G. B.; Konai, M. M.; Uppu, D. S. S. M.; Paramanandham, K.;
Shome, B. R.; Ravikumar, R.; Haldar, J., Arylꢀalkylꢀlysines: Membraneꢀactive small molecules
active against murine model of burn infection. ACS Infectious Diseases 2016, 2 (2), 111ꢀ122.
18)
K.; Shome, B. R.; Haldar, J., ArylꢀAlkylꢀLysines: Agents that kill planktonic cells, persister
cells, biofilms of MRSA and protect mice from skinꢀinfection. PLoS One 2015, 10 (12),
e0144094.
Ghosh, C.; Manjunath, G. B.; Konai, M. M.; Uppu, D. S.; Hoque, J.; Paramanandham,
19)
Ghosh, C.; Manjunath, G. B.; Akkapeddi, P.; Yarlagadda, V.; Hoque, J.; Uppu, D. S.;
Konai, M. M.; Haldar, J., Small molecular antibacterial peptoid mimics: the simpler the better! J.
Med. Chem. 2014, 57 (4), 1428ꢀ1436.
20)
minimum inhibitory concentrations of nylonꢀ3 polymers against E. coli. PLoS One 2014, 9 (8),
e104500.
Choi, H.; Chakraborty, S.; Liu, R.; Gellman, S. H.; Weisshaar, J. C., Medium effects on
22
ACS Paragon Plus Environment

Page 23 of 35
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
21)
Hoque, J.; Konai, M. M.; Gonuguntla, S.; Manjunath, G. B.; Samaddar, S.; Yarlagadda,
V.; Haldar, J., Membrane active small molecules show selective broad spectrum antibacterial
activity with no detectable resistance and eradicate biofilms. J. Med. Chem. 2015, 58 (14), 5486ꢀ
5500.
22)
Uppu, D. S.; Samaddar, S.; Ghosh, C.; Paramanandham, K.; Shome, B. R.; Haldar, J.,
Amide side chain amphiphilic polymers disrupt surface established bacterial bioꢀfilms and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
protect mice from chronic Acinetobacter baumannii infection. Biomaterials 2016, 74, 131ꢀ143.
23)
Latge, J. P., Aspergillus fumigatus and aspergillosis. Clin. Microbiol. Rev. 1999, 12 (2),
310ꢀ350.
24)
Mascio, C. T.; Alder, J. D.; Silverman, J. A., Bactericidal action of daptomycin against
stationaryꢀphase and nondividing Staphylococcus aureus cells. Antimicrob. Agents Chemother.
2007, 51 (12), 4255ꢀ4260.
25)
needs, and new approaches. CSH Perspect. Med. 2014, 4 (5), a019703
26) Ghosh, C.; Haldar, J., Membraneꢀactive small molecules: Designs inspired by
antimicrobial peptides. ChemMedChem 2015, 10 (10), 1606ꢀ1624.
27) Trofa, D.; Gacser, A.; Nosanchuk, J. D., Candida parapsilosis, an emerging fungal
pathogen. Clin. Microbiol. Rev. 2008, 21 (4), 606ꢀ625.
28) Fidel, P. L.; Vazquez, J. A.; Sobel, J. D., Candida glabrata: Review of epidemiology,
Roemer, T.; Krysan, D. J., Antifungal drug development: challenges, unmet clinical
pathogenesis, and clinical disease with comparison to C. albicans. Clin. Microbiol. Rev. 1999, 12
(1), 80ꢀ96.
29)
KwonꢀChung, K. J.; Fraser, J. A.; Doering, T. L.; Wang, Z. A.; Janbon, G.; Idnurm, A.;
Bahn, Y. S., Cryptococcus neoformans and Cryptococcus gattii, the etiologic agents of
cryptococcosis. CSH Perspect. Med. 2014, 4 (7), a019760.
30)
Wang, Q.; Rager, J. D.; Weinstein, K.; Kardos, P. S.; Dobson, G. L.; Li, J.; Hidalgo, I. J.,
Evaluation of the MDRꢀMDCK cell line as a permeability screen for the bloodꢀbrain barrier. Int.
J. Pharm. 2005, 288 (2), 349ꢀ359.
31)
Tsinman, O.; Tsinman, K.; Sun, N.; Avdeef, A., Physicochemical selectivity of the BBB
microenvironment governing passive diffusionꢀꢀmatching with a porcine brain lipid extract
artificial membrane permeability model. Pharm. Res. 2011, 28 (2), 337ꢀ363.
32)
Di, L.; Kerns, E. H.; Bezar, I. F.; Petusky, S. L.; Huang, Y., Comparison of bloodꢀbrain
barrier permeability assays: in situ brain perfusion, MDR1ꢀMDCKII and PAMPAꢀBBB. J.
Pharm. Sci. 2009, 98 (6), 1980ꢀ1991.
33)
34)
http://wwwnc.cdc.gov/eid/article/10/1/03ꢀ0119_article
You, J.; Du, L.; King, J. B.; Hall, B. E.; Cichewicz, R. H., Smallꢀmolecule suppressors of
Candida albicans biofilm formation synergistically enhance the antifungal activity of
amphotericin B against clinical Candida isolates. ACS Chem. Biol. 2013, 8 (4), 840ꢀ848.
35)
Wang, S.; Wang, Y.; Liu, W.; Liu, N.; Zhang, Y.; Dong, G.; Liu, Y.; Li, Z.; He, X.;
Miao, Z.; Yao, J.; Li, J.; Zhang, W.; Sheng, C., Novel carboline derivatives as potent antifungal
lead compounds: design, synthesis, and biological evaluation. ACS Med. Chem. Lett. 2014, 5 (5),
506ꢀ511.
23
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 24 of 35
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1. Antifungal activity of BCK and NCK compounds against Candida albicans and
Cryptococcus neoformans.
Compounds
C. albicans SC5314
C. neoformans H99
MIC
MFC
MIC
MFC
ꢀ1
ꢀ1
ꢀ1
ꢀ1
(µg mL )
(µg mL )
(µg mL )
(µg mL )
NCKꢀ6
NCKꢀ8
>50
>50
25
3.1
3.1
3.1
3.1
6.2
25
3.1
3.1
3.1
3.1
6.2
25
25
NCKꢀ10
BCKꢀ10
BCKꢀ12
BCKꢀ14
12.5
25
25
50
25
25
>50
>50
Results are an average of at least two independent experiments and each experiment was
performed in triplicates.
24
ACS Paragon Plus Environment

Page 25 of 35
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. Activity of NCKꢀ10 against clinical isolates of pathogenic fungi in comparison to
amphotericin B and fluconazole.ꢀ
NCKꢀ10
MFC
Amphotericin B
MIC MFC
Fluconazole
MIC MFC
Fungal strains
MIC
(µg mLꢀ1) (µg mLꢀ1) (µg mLꢀ1) (µg mLꢀ1) (µg mLꢀ1) (µg mLꢀ1)
Candida albicans
NRꢀ29448
Candida albicans
NRꢀ29351
Candida albicans
NR29438
Candida albicans
NRꢀ29366
Candida albicans
NRꢀ29367
Candida albicans
ATCC 26790
Candida albicans
NRꢀ29365
Candida albicans
ATCC MYA573
Candida albicans
ATCC 64124
0.5
1
1
0.5
0.5
1
1
>64
0.5
>64
>64
>64
>64
0.5
>64
>64
2
ND*
8
2
0.5
2
2
2
ND
ND
ND
ND
>64
ND
ND
2
2
2
1
1
2
2
1
2
2
2
1
1
0.5
0.5
2
0.5
0.5
2
2
2
1
1
1
1
Candida parapbsilosis 0.5
ATCC22019
Candida glabrata
1
0.5
1
2
1
1
1
32
>64
ATCC MYAꢀ2950
25
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 26 of 35
1
2
3
4
5
6
7
8
9
Candida glabrata
ATCC 66032
Candida tropicalis
ATCC 1369
Candida tropicalis
ATCC 13803
Cryptococcus gattii
NRꢀ43208
Cryptococcus gattii
NRꢀ43209
Cryptococcus
neoformans NR41294
Cryptococcus
neoformans
NRꢀ41296
Cryptococcus
neoformans
NRꢀ41295
Cryptococcus
neoformans
NRꢀ41292
Aspergillus fumigatus
NRꢀ35308
Aspergillus fumigatus
NRꢀ35304
Aspergillus fumigatus
NRꢀ35303
Aspergillus fumigatus
NRꢀ35301
Aspergillus fumigatus
1
1
0.5
0.5
0.5
1
2
16
2
>64
>64
>64
2
0.5
1
0.5
1
0.5
2
0.5
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
1
2
8
8
1
1
1
4
4
8
2
2
1
1
4
8
2
2
1
1
16
8
32
8
2
1
2
1
0.5
1
0.5
16
>64
>64
>64
>64
>64
ND
4
4
4
4
2
4
4
4
4
2
0.25
0.025
≤0.0625 0.25
≤0.0625 0.125
ND
ND
ND
ND*
0.25
0.5
0.25
0.5
NRꢀ35302
*ND stands for not detectable
26
ACS Paragon Plus Environment

Page 27 of 35
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Effective permeability coefficient of NCKꢀ10 and control drugs obtained from the
PAMPAꢀBBB assay.
Test Agent
NCKꢀ10
Atenolol
Mean Pe (cm/sec)
2.87 × 10ꢀ6
Inference
Good permeability
Low permeability control
0.000013 × 10ꢀ6
Verapamil
14.6 × 10ꢀ6
High permeability control
27
ACS Paragon Plus Environment

ACS Infectious Diseases
Page 28 of 35
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Chemical structures of compounds presented in this study.
28
ACS Paragon Plus Environment

Page 29 of 35
ACS Infectious Diseases
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Kinetics of fungicidal action of NCKꢀ10.
29
ACS Paragon Plus Environment