ML212: A small-molecule probe for investigating fluconazole resistance mechanisms in Candida albicans

Article abstract of DOI:10.3762/bjoc.9.171

The National Institutes of Health Molecular Libraries and Probe Production Centers Network (NIH-MLPCN) screened >300,000 compounds to evaluate their ability to restore fluconazole susceptibility in resistant Candida albicans isolates. Additional counter screens were incorporated to remove substances inherently toxic to either mammalian or fungal cells. A substituted indazole possessing the desired bioactivity profile was selected for further development, and initial investigation of structure-activity relationships led to the discovery of ML212.

Full text of DOI:10.3762/bjoc.9.171

ML212: A small-molecule probe for investigating
fluconazole resistance mechanisms
in Candida albicans
Willmen Youngsaye1, Cathy L. Hartland1, Barbara J. Morgan1, Amal Ting1,
Partha P. Nag1, Benjamin Vincent2,3, Carrie A. Mosher1, Joshua A. Bittker1,
Sivaraman Dandapani1, Michelle Palmer1, Luke Whitesell2,
Susan Lindquist2,4, Stuart L. Schreiber1,5 and Benito Munoz*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 1501–1507.
1Chemical Biology Platform and Probe Development Center, Broad
Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA
02142, USA, 2Whitehead Institute for Biomedical Research, 9
Cambridge Center, Cambridge, MA 02142, USA, 3Microbiology
Graduate Program, Massachusetts Institute of Technology, 77
Massachusetts Avenue, Cambridge, MA 02139, USA, 4Department of
Biology and Howard Hughes Medical Institute, Massachusetts
Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA
02139, USA and 5Howard Hughes Medical Institute, Broad Institute of
Harvard and MIT, 7 Cambridge Center, Cambridge, MA 02142, USA
doi:10.3762/bjoc.9.171
Received: 12 March 2013
Accepted: 10 June 2013
Published: 26 July 2013
This article is part of the Thematic Series "Synthetic probes for the study
of biological function".
Guest Editor: J. Aubé
Email:
© 2013 Youngsaye et al; licensee Beilstein-Institut.
License and terms: see end of document.
Benito Munoz* - bmunoz@broadinstitute.org
* Corresponding author
Keywords:
antifungal; Candida albicans; chemosensitizer; fluconazole; Molecular
Libraries Probe Production Center Network (MLPCN)
Abstract
The National Institutes of Health Molecular Libraries and Probe Production Centers Network (NIH-MLPCN) screened >300,000
compounds to evaluate their ability to restore fluconazole susceptibility in resistant Candida albicans isolates. Additional counter
screens were incorporated to remove substances inherently toxic to either mammalian or fungal cells. A substituted indazole
possessing the desired bioactivity profile was selected for further development, and initial investigation of structure–activity rela-
tionships led to the discovery of ML212.
Introduction
Discovery of antimicrobial agents possessing unique structural Chemosensitization of resistant organisms is a complementary
motifs or a novel mechanism of action is critical to counter and approach that capitalizes upon the existing arsenal of antimicro-
control the rising incidence of drug-resistant pathogens [1-6]. bials to combat this medical dilemma [7-10]. By undermining
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the resistance mechanisms of the target pathogen, it is possible surmounting this inherent resistance [13-16]. The screen was
to restore efficacy to previously ineffective drugs thereby conducted by using a cell-based assay with integrated counter
prolonging their status as frontline treatments. This, in turn, screens to remove compounds acting through previously estab-
affords critical lead-time towards the development of novel lished methods for overturning drug resistance in C. albicans.
antimicrobial drugs.
In addition, substances intrinsically toxic to either mammalian
or fungal cells were eliminated.
The National Institutes of Health Molecular Libraries and Probe
Production Centers Network (NIH-MLPCN) recently
Results and Discussion
performed a high-throughput screening (HTS) campaign to Screening results
search for potential chemosensitizers of the pathogenic fungus In order to identify nonfungitoxic chemosensitizers of Candida
Candida albicans [11]. The C. albicans clinical isolates used in albicans, compounds from the NIH’s Molecular Libraries Small
this study demonstrate a range of resistance to the widely Molecule Repository (MLSMR) [17] were evaluated in the
prescribed triazole antimycotic fluconazole (Flc) [12], and the screening cascade summarized in Figure 1. The C. albicans
objective was to identify novel small molecules capable of strains used in the primary screen and secondary assay 1
Figure 1: Assay pipeline for triaging hits. Individual assay cut-offs are given in italics.
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(CaCi-2 and CaCi-8, respectively) are both clinical isolates that more in the entire assay tree outlined previously in Figure 1.
partially respond to fluconazole treatment [12]. The minimum Following this re-evaluation, methyl 3-phenyl-1H-indazole
inhibitory concentration (MIC) of fluconazole against CaCi-2 acetate (1, Figure 2) emerged as an attractive candidate for
and CaCi-8 was reported to be 2 µg/mL and 8 µg/mL, respect- further development. A commercial sample of compound 1
ively [12]. However in our hands, these strains continue to shows good activity against CaCi-2 and CaCi-8 (IC50 = 0.8 and
proliferate steadily (albeit at a reduced rate) when treated with 2.3 μM, respectively) with no apparent effect on 3T3 fibro-
fluconazole at or above the reported MIC. This behavior may blasts (IC50 > 26 μM). Compound 1 also possesses good solu-
contribute to the inability of fluconazole therapy to effectively bility in PBS (79 μM) and is synthetically tractable with several
clear the infection and allows for the further development of points of diversity readily accessible. Consequently, compound
resistance [12].
1 and a collection of closely related analogues were prepared to
enable investigation of possible structure–activity relationships
A total of 302,509 compounds from the MLSMR were tested at (SAR) [18]. In addition to indazole 1, two other structurally
7.5 µM for their ability to inhibit completely the growth of distinct scaffolds (2 and 3) were also selected for follow-up
CaCi-2 cells that are concurrently treated with 8 µg/mL studies and those works are communicated elsewhere [19,20].
fluconazole (Figure 1). With a minimum requirement of 75%
inhibition, 1893 actives were recorded, corresponding to an
overall hit rate of 0.6%. Of the active compounds, 1654 were
available for retesting in a dose-response format. This subset
was resubjected to the primary assay and was also tested
concurrently in two secondary assays. Secondary assay 1
measured chemosensitization of the more resistant CaCi-8
strain. Secondary assay 2 eliminated anything with inherently
antifungal activity. These three assays cooperatively removed
almost 80% of the original hits, leaving 350 candidates. A final
assay (secondary assay 3) was incorporated to discard any com-
pound displaying toxicity to mammalian fibroblasts. The fibro-
blast toxicity assay removed another 54 compounds to leave a
Figure 2: Hit compounds selected for further optimization.
total of 296 hits.
From the 296 hits remaining, 29 were selected for revalidation Chemistry
from dry powders obtained from commercial vendors or resyn- Two different routes were adopted to access the various func-
thesis. Once their identity and purity was ascertained by LCMS tionalized indazoles required to evaluate the SAR associated
and 1H NMR analysis, these 29 candidates were tested once with hit compound 1 (Scheme 1). The robust Suzuki–Miyaura
Scheme 1: Preparation of substituted methyl 2-(1H-indazol-1-yl)acetates. Reagents and conditions: (a) Et3N, Boc2O, DMAP, tetrahydrofuran;
(b) ArB(OH)2, Pd(PPh3)4, aqueous Na2CO3, 1,4-dioxane, 120 °C; (c) methyl bromoacetate, K2CO3, acetone, 60 °C; (d) alkylmagnesium bromide,
Et2O, 0 °C; (e) Dess–Martin periodinane, CH2Cl2; (f) hydrazine hydrate, 175 °C.
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reaction was selected for the preparation of analogues bearing
substituents around the central indazole core. This approach
also permitted rapid replacement of the phenyl ring at C3 with
functionalized phenyl rings and alternative heterocycles.
Preliminary attempts to couple substituted 3-iodoindazoles 4
failed to produce isolable amounts of the desired product
directly. Subsequently, the indazoles were protected as their
tert-butyl carbamates 5 prior to undergoing palladium-medi-
ated Suzuki reactions with various boronic acid partners. Under
the reaction conditions, the carbamate-protecting group was
also cleaved to afford the desired 3-arylindazoles, albeit as the
unprotected systems 6. Alkylation with methyl bromoacetate
and potassium carbonate in hot acetone completed the syn-
thesis of compounds 7.
Table 1: Activity of substituted indazole cores.a
Cpd
R
CaCi-2
CaCi-8
IC50 (µM)b IC50 (µM)b
1
H
2.2 ± 1.0
5.2 ± 2.2
3.5 ± 1.9
8.2 ± 3.2
11
12
13
14
15
16
17
18
19
5-Me
5-OMe
5-F
14.9 ± 5.7 22.1 ± 6.2
11.2 ± 7.2 inactive
5-Cl
inactive
5.9 ± 3.1
9.5 ± 2.5
4.0 ± 1.3
inactive
inactive
inactive
inactive
12.4 ± 0.5
8.4 ± 0.5
inactive
inactive
6-Me
6-OMe
6-Cl
In order to prepare 3-alkylindazoles, 2-fluorobenzaldehyde was
first treated with alkylmagnesium bromides, and the resulting
benzyl alcohols were immediately oxidized with Dess–Martin
reagent. Alkyl phenyl ketones 9 and hydrazine hydrate were
then reacted under microwave conditions to assemble the inda-
zole ring 10. The ester side chain of 11 was installed under the
same conditions described above for the alkylation of 3-arylin-
dazoles.
6-CF3
7-CF3
20
inactive
inactive
aCaCi-2 and CaCi-8 cells were incubated at 37 °C for 48 hours with
test compound and 8 µg/mL (26 µM) fluconazole. bAverage of at least
three independent experiments, performed in duplicate. Inactive com-
pounds displayed negligible activity at concentrations below 26 µM.
In vitro activity and SAR
All of the compounds prepared above were evaluated for their
ability to chemosensitize the C. albicans test strains CaCi-2 and
CaCi-8 towards fluconazole. As described above, the fungi
were simultaneously incubated with test compounds and
fluconazole (8 µg/mL) for 48 hours to determine if any combi-
nations could fully inhibit fungal growth. The DMSO/flucona-
zole combination served as an internal control.
inactive analogue 21. Similarly, installing acyclic alkyl systems
such as an ethyl (22) or tert-butyl group (23) does not yield
active compounds. Proceeding to cycloalkanes, a SAR trend
begins to emerge. With the smallest cycloalkane replacement,
the cyclopropyl derivative 24 is inactive whereas the larger
cyclohexane of 25 yields an active chemosensitizer
(IC50 = 2.3 μM). When alternative heteroaromatic rings (26–29)
were prepared, none of the examined systems possess any
significant cellular activity.
Substitution of the indazole core generally leads to a potency
reduction to varying degrees (Table 1). The 5- and 6-positions
can accommodate smaller functionalities such as methyl or
chloro groups (11, 15, and 17) and still retain modest efficacy.
The inactivity of 14 compared to 17 suggests that the 6-pos-
ition may occupy a slightly larger pocket. Electron-with- One possible explanation for the observed activity is that the
drawing substituents (13, 18, and 19) appear detrimental to binding pocket includes a small cleft that is best occupied by
activity regardless of their position, while the weak potency of flat structures. Compounds 21, 22, and 24 are presumably too
the methoxy compounds (12 and 16) suggests that there may be small to fit properly into this crevice, while the tert-butyl
a limit to how far the western region may be extended. derivative 23 may simply be too large. However, the inactivity
Pyridylindazole 20, wherein a nitrogen atom is inserted in the of the heteroaromatic counterparts 26–29 may imply that more
place of a carbon, displays no cellular activity. Based on these than a steric constraint is operative within this putative groove;
preliminary results, a more extensive SAR investigation of this a possible electronic requirement for this substituent may exist
region was postponed in order to explore other regions of the as well.
scaffold.
Returning to the original phenyl ring, various substituents were
We proceeded to evaluate the SAR of the C3 substituent next introduced at different positions about the ring to probe for
(Table 2). Removal of the original benzene ring produces the further electronic and steric effects associated with this region
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Table 2: Investigation of 3-substituted indazoles.a
Table 3: Substituent effects associated with the 3-phenyl ring.a
Cpd
R
CaCi-2
CaCi-8
IC50 (µM)b
IC50 (µM)b
Cpd
R
CaCi-2
IC50 (µM)b
CaCi-8
IC50 (µM)b
21
22
-H
inactive
inactive
inactive
inactive
-Et
1
H
2.2 ± 1.0
4.8 ± 2.5
4.2 ± 1.7
8.3 ± 2.8
inactive
3.5 ± 1.9
19.5 ± 14.2
14.1 ± 6.4
12.3 ± 3.9
inactive
23
24
25
inactive
inactive
2.3 ± 0.3
inactive
inactive
6.0 ± 1.7
30
31
32
33
34
35
36
37
38
39
40
41
4′-Me
4′-OMe
4′-F
4′-CF3
4′-CN
3′-Me
inactive
20.5 ± 0.3
1.9 ± 0.9
1.5 ± 0.6
2.0 ± 0.2
4.2 ± 4.1
7.8 ± 2.6
11.4 ± 1.7
inactive
1.1 ± 0.6
0.7 ± 0.3
0.8 ± 0.1
1.7 ± 0.4
5.3 ± 0.5
9.3 ± 3.6
inactive
3′-OMe
3′-NMe2
3′-F
26
27
inactive
inactive
inactive
2′-Me
2′-OMe
3′,5′-di-OMe
21.1 ± 3.3
aCaCi-2 and CaCi-8 cells were incubated at 37 °C for 48 hours with
test compound and 8 µg/mL (26 µM) fluconazole. bAverage of at least
three independent experiments, performed in duplicate. Inactive com-
pounds displayed negligible activity at concentrations below 26 µM.
28
29
inactive
inactive
inactive
(IC50 > 26 µM). The activity of p-tolyl 30 and p-anisoyl 31
indicates that the inefficacy of 33 and 34 cannot be solely attrib-
uted to steric considerations. The inactivity of the 3,5-
dimethoxyphenyl ring (41) suggests the indazole’s C3
substituent may reside in an asymmetric pocket.
12.9 ± 1.7
aCaCi-2 and CaCi-8 cells were incubated at 37 °C for 48 hours with
test compound and 8 µg/mL (26 µM) fluconazole. bAverage of at least
three independent experiments, performed in duplicate. Inactive com-
pounds displayed negligible activity at concentrations below 26 µM.
As the most potent chemosensitizer of both C. albicans CaCi-2
(Table 3). With regards to steric requirements, it appears that and CaCi-8 (IC50 = 0.7 and 1.5 µM, respectively) with good
neither the para- (compounds 30–34) nor ortho-positions (com- solubility (55 µM in PBS), compound 36 was nominated as
pounds 39 and 40) are particularly amenable to functionaliza- MLPCN probe ML212. Exposure to human or murine plasma
tion. While bioactivity is still observed with these substances, revealed significant chemical instability (<10% remaining after
they all appear to be only weakly active (IC50 = 2–8 μM). 5 h incubation), and this is attributed to the ester hydrolysis of
Conversely, the meta-substituted systems 35–38 prove to be the the side chain. Follow-up studies investigating the SAR of the
most conducive to potency. The methyl ether 36 is potent side chain and addressing this liability will be reported shortly.
against CaCi-2 (IC50 = 0.7 μM) as is the N,N-dimethylamine Additional profiling of ML212 determined that the probe is
variant (37, IC50 = 0.8 μM). The weakest compound of this nontoxic to C. albicans in the absence of fluconazole
series, 3-fluoro derivative 38, still shows low micromolar (IC50 > 26 µM after 48 h incubation), neither does it show any
activity (IC50 = 1.7 μM). The weak activity of 38 may be tied to toxicity towards murine 3T3 fibroblasts (IC50 > 26 µM).
the electronegativity of fluorine. This is best illustrated with Hsp90-dependent and calcineurin-dependent signaling path-
compounds 32–34. While the 4-fluoro analogue is a weak ways have been previously implicated in maintaining flucona-
chemosensitizer of CaCi-2 and CaCi-8, introducing more elec- zole resistance in C. albicans [21], but ML212 does not inhibit
tron-withdrawing substituents, such as trifluoromethyl (33) these pathways in yeast-reporter assays (IC50 > 26 µM). Identi-
and cyano (34) groups, results in complete inactivity fication of ML212’s molecular target is ongoing, as well as
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4. Vandeputte, P.; Ferrari, S.; Coste, A. T. Int. J. Microbiol. 2012,
No. 713687. doi:10.1155/2012/713687
efforts to determine the efficacy of ML212 against diverse
mechanisms of fluconazole resistance, including biofilm forma-
tion, drug-target mutations, and efflux-pump amplification.
5. Pfaller, M. A.; Diekema, D. J. Clin. Microbiol. Rev. 2007, 20, 133–163.
doi:10.1128/CMR.00029-06
6. Sanglard, D.; Odds, F. C. Lancet Infect. Dis. 2002, 2, 73–85.
doi:10.1016/S1473-3099(02)00181-0
Conclusion
7. Lavigne, J.-P.; Brunel, J.-M.; Chevalier, J.; Pagès, J.-M.
J. Antimicrob. Chemother. 2010, 65, 799–801. doi:10.1093/jac/dkq031
8. Gallo, S.; Chevalier, J.; Mahamoud, A.; Eyraud, A.; Pagès, J.-M.;
Barbe, J. Int. J. Antimicrob. Agents 2003, 22, 270–273.
doi:10.1016/S0924-8579(03)00215-2
High-throughput screening of 300,000 compounds from the
NIH’s MLSMR collection identified several substances that
potentiate the effect of fluconazole in fluconazole-resistant
Candida albicans clinical isolates. Among the numerous hits,
3-phenylindazole 1 was selected for chemical optimization,
resulting in the identification of 3-(3-anisoyl)indazole 36 as new
small-molecule probe (ML212) to facilitate investigation of the
various mechanisms used by C. albicans to withstand flucona-
zole. Elucidation of ML212’s mechanism of action may afford
new targets to exploit in the continuing efforts to develop novel
antimycotics and combat increasingly prevalent drug-resistance.
Samples of ML212 are available free of charge, on request.
9. Kim, J.; Campbell, B.; Mahoney, N.; Chan, K.; Molyneux, R.; May, G.
Biochem. Biophys. Res. Commun. 2008, 372, 266–271.
doi:10.1016/j.bbrc.2008.05.030
10.Cernicka, J.; Kozovska, Z.; Hnatova, M.; Valachovic, M.; Hapala, I.;
Riedl, Z.; Hajós, G.; Subik, J. Int. J. Antimicrob. Agents 2007, 29,
170–178. doi:10.1016/j.ijantimicag.2006.08.037
11.The complete results of the HTS assay can be viewed online free of
charge on PubChem.
http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1979.
12.Redding, S.; Smith, J.; Farinacci, G.; Rinaldi, M.; Fothergill, A.;
Rhine-Chalberg, J.; Pfaller, M. Clin. Infect. Dis. 1994, 18, 240–242.
doi:10.1093/clinids/18.2.240
Supporting Information
13.DiGirolamo, J. A.; Li, X.-C.; Jacob, M. R.; Clark, A. M.; Ferreira, D.
J. Nat. Prod. 2009, 72, 1524–1528. doi:10.1021/np900177m
14.Gamarra, S.; Rocha, E. M. F.; Zhang, Y.-Q.; Park, S.; Rao, R.;
Perlin, D. S. Antimicrob. Agents Chemother. 2010, 54, 1753–1761.
doi:10.1128/AAC.01728-09
Detailed experimental protocols for cellular assays and for
the preparation of representative compounds 25 and 36 are
provided. Proton NMR spectra for all prepared compounds
are also available.
15.Guo, X.-L.; Leng, P.; Yang, Y.; Yu, L.-G.; Lou, H.-X. J. Appl. Microbiol.
2008, 104, 831–838. doi:10.1111/j.1365-2672.2007.03617.x
16.Mai, A.; Rotili, D.; Massa, S.; Brosch, G.; Simonetti, G.; Passariello, C.;
Palamara, A. T. Bioorg. Med. Chem. Lett. 2007, 17, 1221–1225.
doi:10.1016/j.bmcl.2006.12.028
Supporting Information File 1
Detailed assay protocols and compound synthesis.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-9-171-S1.pdf]
17.Additional information about the MLSMR can be found online at
http://mli.nih.gov/mli/secondary-menu/mlscn/ml-small-molecule-reposit
ory/
Supporting Information File 2
18.The Present Communication details a portion of the work previously
described in official Probe Report submitted to the NIH upon project
completion. The official Probe Report has been made available online
by the NIH, free of charge: Hartland, C. L.; Youngsaye, W.; Morgan, B.;
Ting, A.; Nag, P.; Burhlage, S.; Johnston, S.; Bittker, J.; Vincent, B.;
Whitesell, L.; Dandapani, S.; MacPherson, L.; Munoz, B.; Palmer, M.;
Lindquist, S.; Schreiber, S. L. “Identification of small molecules that
selectively inhibit fluconazole-resistant Candida albicans in the
presence of fluconazole but not in its absence - Probe 2”. In: Probe
Reports from the NIH Molecular Libraries Program [Internet]. Bethesda
(MD): National Center for Biotechnology Information (US); 2010-.
Available from: http://www.ncbi.nlm.nih.gov/books/NBK98920/
19.Youngsaye, W.; Vincent, B.; Hartland, C. L.; Morgan, B. J.;
Buhrlage, S. J.; Johnston, S.; Bittker, J. A.; MacPherson, L.;
Dandapani, S.; Palmer, M.; Whitesell, L.; Lindquist, S.; Schreiber, S. L.;
Munoz, B. Bioorg. Med. Chem. Lett. 2011, 21, 5502–5505.
doi:10.1016/j.bmcl.2011.06.105
NMR spectra of reported compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-9-171-S2.pdf]
Acknowledgements
This work was funded by the NIH’s MLPCN program (1 U54
HG005032-1 awarded to S.L.S.). BV, LW, and SL are grateful
for funding from the NIH (1 R03 MH086456-01). The authors
are grateful to Dr. Spencer Redding (University of Texas Health
Science Center San Antonio) for graciously providing samples
of C. albicans CaCi-2 and CaCi-8.
References
1. Martínez, J. L. Future Med. Chem. 2012, 4, 347–359.
20.Youngsaye, W.; Dockendorff, C.; Vincent, B.; Hartland, C. L.;
Bittker, J. A.; Dandapani, S.; Palmer, M.; Whitesell, L.; Lindquist, S.;
Schreiber, S. L.; Munoz, B. Bioorg. Med. Chem. Lett. 2012, 22,
3362–3365. doi:10.1016/j.bmcl.2012.02.035
doi:10.4155/fmc.12.2
2. Hawkey, P. M.; Jones, A. M. J. Antimicrob. Chemother. 2009, 64
(Suppl. 1), i3–i10. doi:10.1093/jac/dkp256
21.Cowen, L. E.; Lindquist, S. Science 2005, 309, 2185–2189.
doi:10.1126/science.1118370
3. Gould, I. M. Int. J. Antimicrob. Agents 2008, 32 (Suppl. 1), S2–S9.
doi:10.1016/j.ijantimicag.2008.06.016
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