Drugging the Folate Pathway in Mycobacterium tuberculosis: The Role of Multi-targeting Agents

Article abstract of DOI:10.1016/j.chembiol.2019.02.013

The folate biosynthetic pathway offers many druggable targets that have yet to be exploited in tuberculosis therapy. Herein, we have identified a series of small molecules that interrupt Mycobacterium tuberculosis (Mtb) folate metabolism by dual targeting of dihydrofolate reductase (DHFR), a key enzyme in the folate pathway, and its functional analog, Rv2671. We have also compared the antifolate activity of these compounds with that of para-aminosalicylic acid (PAS). We found that the bioactive metabolite of PAS, in addition to previously reported activity against DHFR, inhibits flavin-dependent thymidylate synthase in Mtb, suggesting a multi-targeted mechanism of action for this drug. Finally, we have shown that antifolate treatment in Mtb decreases the production of mycolic acids, most likely due to perturbation of the activated methyl cycle. We conclude that multi-targeting of the folate pathway in Mtb is associated with highly potent anti-mycobacterial activity. Hajian et al. have investigated the inhibitory activity of a series of small-molecule antifolates and para-aminosalicylic acid against Mycobacterium tuberculosis, the causative agent of tuberculosis, and show that these compounds exert their activity by acting on multiple targets within the folate biosynthetic pathway.

Full text of DOI:10.1016/j.chembiol.2019.02.013

Article
Drugging the Folate Pathway in Mycobacterium
tuberculosis: The Role of Multi-targeting Agents
Graphical Abstract
Authors
Behnoush Hajian, Eric Scocchera,
´
´
Carolyn Shoen, ..., Jana Kordulakova,
Michael Cynamon, Dennis Wright
Correspondence
dennis.wright@uconn.edu
In Brief
Hajian et al. have investigated the
inhibitory activity of a series of small-
molecule antifolates and para-
aminosalicylic acid against
Mycobacterium tuberculosis, the
causative agent of tuberculosis, and
show that these compounds exert their
activity by acting on multiple targets
within the folate biosynthetic pathway.
Highlights
d
d
d
d
Inhibitors of the folate pathway in Mycobacterium
tuberculosis have been identified
These molecules are dual inhibitors of dihydrofolate
reductase and Rv2671
para-Aminosalicylic acid metabolite inhibits flavin-
dependent thymidylate synthase
Antifolates decrease the level of precursors of mycolic acids
in M. tuberculosis
Hajian et al., 2019, Cell Chemical Biology 26, 1–11
June 20, 2019 Published by Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2019.02.013

Please cite this article in press as: Hajian et al., Drugging the Folate Pathway in Mycobacterium tuberculosis: The Role of Multi-targeting Agents, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.013
Cell Chemical Biology
Article
Drugging the Folate Pathway
in Mycobacterium tuberculosis: The Role
of Multi-targeting Agents
Behnoush Hajian,1 Eric Scocchera,1 Carolyn Shoen,2 Jolanta Krucinska,1 Kishore Viswanathan,1
1
3
1
4
4
ꢀ
´
´
´
Narendran G-Dayanandan, Heidi Erlandsen, Alexavier Estrada, Katarı´na Mikusova, Jana Kordulakova,
Michael Cynamon,2 and Dennis Wright1,5,
*
1Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269, USA
2Syracuse VA Medical Center, Syracuse, NY 13210, USA
3Center for Open Research Resources and Equipment, University of Connecticut, Storrs, CT 06269, USA
4
ꢀ
Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynska Dolina CH-1, Ilkovicova 6, 842 15,
´
Bratislava, Slovakia
5Lead Contact
*Correspondence: dennis.wright@uconn.edu
https://doi.org/10.1016/j.chembiol.2019.02.013
SUMMARY
or antifolates (Kumar et al., 2015; Nixon et al., 2014). Antifolates
interrupt the production of reduced folate cofactors by inhibit-
The folate biosynthetic pathway offers many drug- ing key enzymes in the folate metabolic pathway (Figure 1)
(Scocchera and Wright, 2017). Synthesis and recycling of
reduced folates is essential for the de novo synthesis of methi-
gable targets that have yet to be exploited in tubercu-
losis therapy. Herein, we have identified a series of
small molecules that interrupt Mycobacterium tuber-
culosis (Mtb) folate metabolism by dual targeting
of dihydrofolate reductase (DHFR), a key enzyme
in the folate pathway, and its functional analog,
Rv2671. We have also compared the antifolate
activity of these compounds with that of para-amino-
onine, purines, and deoxythymidine monophosphate (dTMP). A
lynchpin in the folate pathway, dihydrofolate reductase (DHFR)
(encoded by folA), catalyzes the reduction of dihydrofolate
(DHF) to tetrahydrofolate (THF) using NADPH as an electron
donor. Due to its pivotal role in nucleic acid synthesis, and
high conservation through evolution, DHFR has served as an
ideal antiproliferative drug target for cancer and infectious dis-
eases (Visentin et al., 2012). Despite the success of antifolates
as anticancer and antimicrobial agents, they are an underused
salicylic acid (PAS). We found that the bioactive
metabolite of PAS, in addition to previously reported
activity against DHFR, inhibits flavin-dependent thy- drug class in TB therapy, particularly those targeting DHFR.
Current DHFR inhibitors either show poor potency against
Mtb DHFR (e.g., trimethoprim) or fail to inhibit the growth of
midylate synthase in Mtb, suggesting a multi-tar-
geted mechanism of action for this drug. Finally, we
have shown that antifolate treatment in Mtb de-
creases the production of mycolic acids, most likely
due to perturbation of the activated methyl cycle. We
conclude that multi-targeting of the folate pathway in
Mtb is associated with highly potent anti-mycobac-
terial activity.
live Mtb despite good enzyme potency, most likely due to
lack of permeability (e.g., methotrexate [MTX], pyrimethamine,
and trimetrexate) (Nixon et al., 2014).
Previously, we have reported the antimycobacterial activity of
a series of small-molecule antifolates that are designed to
inhibit DHFR enzymes of various microorganism (Hajian et al.,
2016). Screening of our compound library against wild-type
and multidrug-resistant Mtb strains identified a chemically
distinct series that we have termed ionized non-classical antifo-
INTRODUCTION
lates (INCAs) (Figure 2) with remarkable activity at both the
target and organism levels. INCAs enter cells through passive
Tuberculosis (TB) has traveled with humans since the dawn of diffusion and are characterized by the presence of a single
our species and has claimed millions of lives throughout human acidic functionality which affords ionic character at all pHs,
history. TB, caused by Mycobacterium tuberculosis (Mtb), is the either as a zwitterion or with net single negative charge (ꢀ1).
leading cause of death from a single infectious agent. Despite This physico-chemical profile stands in contrast to both lipo-
more than half a century of anti-TB chemotherapy, the onerous philic antifolates, which are neutral or positively charged, and
drug regimens have led to poor adherence, treatment failure, classical antifolates––such as MTX––which carry a double-
and the growing frequency of drug-resistant TB. After decades negative charge and, therefore, must be actively transported
of relative inactivity in TB drug discovery, the pressing need for into cells.
novel therapeutics that can tackle these issues has renewed
research interest in this area (Koul et al., 2011).
In this work, we describe in-depth mechanistic studies of
select INCA inhibitors and compare their anti-DHFR activity
One class of therapeutics that have recently gained interest with that of para-aminosalicylic acid (PAS), the only antifolate
for their antitubercular activity are the folate antimetabolites, currently used in TB therapy. PAS, one of the few drugs available
Cell Chemical Biology 26, 1–11, June 20, 2019 Published by Elsevier Ltd.
1

Please cite this article in press as: Hajian et al., Drugging the Folate Pathway in Mycobacterium tuberculosis: The Role of Multi-targeting Agents, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.013
Figure 1. Mycobacterium tuberculosis Folate Pathway
Canonical pathway enzyme (green): DHPS, dihydropteroate synthase; DHFS, dihydrofolate synthase; DHFR, dihydrofolate reductase; TS, thymidylate synthase;
SHMT, serine hydroxyl methyltransferase. Alternate pathway enzymes (orange): FDTS, flavin-dependent thymidylate synthase, Rv2671.
for treatment of drug-resistant TB, has been recently shown to RESULTS
act as an alternative substrate for folate biosynthesis in Mtb,
and the product of its metabolism has demonstrated inhibitory Kinetic Profiling of INCAs and PAS-M as DHFR Inhibitors
activity against Mtb DHFR (Chakraborty et al., 2013; Dawadi INCAs feature a 2,4-diaminopyrimdine ring linked to a biaryl sys-
et al., 2017; Zheng et al., 2013).
tem through a propargyl bridge. The compounds chosen for this
We have also investigated the effect of these compounds on study included: UCP1172 and UCP1175, representing the INCA
an alternate folate pathway present in Mtb. This alternate class, UCP1063 (a previous generation non-INCA), and PAS-M,
mechanism uses recently discovered enzymes Rv2671 (a sec- which was synthesized through modification of a previously
ond DHFR found in Mtb) and flavin-dependent thymidylate syn- reported procedure (Scheme S1) (Dawadi et al., 2017).
thase (FDTS) (encoded by thyX) to generate THF and dTMP
To determine the relative affinity of these compounds for Mtb
(Figure 1) (Cheng and Sacchettini, 2016; Mishanina et al., DHFR, the half maximal inhibitory concentrations (IC50) were
2016; Zheng et al., 2013). The discovery of these enzymes measured using standard procedures that spectroscopically
has profound implications for antifolate drug discovery as follow the oxidation of NADPH at 340 nm. The inhibitory con-
they provide the organism with a source of facile resistance stant, Ki, was determined from IC50 values using the Cheng-
to antifolates. Overexpression of Rv2671 or thyX has been Prussof equation and Km values (Table S1). MTX, a potent
shown to be associated with resistance to DHFR inhibitors DHFR inhibitor among various species, was used as the positive
and PAS (Moradigaravand et al., 2016; Zhang et al., 2015; control. INCAs and MTX showed potent inhibition of DHFR, with
Zheng et al., 2013). However, it is not clear if the inhibition of Ki values below 10 nM (Table 1). However, PAS-M activity
these compensatory enzymes or multiple inhibition of both against Mtb DHFR was moderate (Ki = 750 nM) compared with
pathways improves the potency of antimycobacterial com- INCAs and MTX. We also tested the inhibitory effect of PAS-M
pounds against the live bacilli.
against Mtb DHFR under various incubation times, protein con-
Here, we show that select INCAs are potent dual inhibitors centrations, and temperatures, with no significant improvement
of both Mtb DHFR and Rv2671 and this dual inhibition contrib- detected (data not shown).
utes to improved potency against Mtb cells. The INCA inhibi-
Puzzled by PAS-M’s low affinity for Mtb DHFR, and to obtain
tors exhibit a slow dissociation rate from DHFR, resulting in a a better measurement of the mode of action of the inhibitors,
long target occupancy, a desirable feature for antibacterials. we determined the dissociation rate of PAS-M and INCAs
PAS metabolite (PAS-M) was found to be a moderate Mtb from Mtb DHFR. It has been shown that drug dissociation
DHFR when compared with INCAs with no significant inhibi- from its target is a critical factor for sustained drug efficacy
tory activity against Mtb Rv2671. Using X-ray crystallography, in vitro and in vivo (Copeland, 2010; Walkup et al., 2015).
we have elucidated drug-target interactions that impart the Slow dissociation of a drug-target complex extends the dura-
potency of INCAs and PAS-M. Further investigations led us tion of drug-target occupancy, or drug-target residence time
to identify FDTS as a critical secondary target for PAS-M sug- (tR), which can be used as a parameter to quantify and
gesting
a multi-targeted mechanism of action for PAS. compare the lifetime of drug-target interactions. Such parame-
Finally, we show that treatment of Mtb cells with INCAs or ters provide additional measurements of drug potency that may
PAS leads to critical disruption of mycolic acids synthesis. not be evident by thermodynamic parameters such as IC50 and
Overall, our findings suggest that multi-targeted antifolates Ki (Tonge, 2017).
are promising candidates for development of new antituber-
cular agents.
The dissociation rates of the inhibitors were determined using
a jump-dilution assay to determine whether PAS-M or INCAs
2
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Figure 2. Structure of INCAs, UCP1063,
Methotrexate (MTX), and PAS-M
Thermal Stability Analysis of Mtb
DHFR Bound to the Inhibitors
We evaluated the thermal stability of Mtb
DHFR upon ligand binding using differen-
tial scanning calorimetry (DSC). It is known
that ligand binding to a protein yields a
complex with increased thermodynamic
stability compared with the unbound pro-
tein. This difference in stability leads to a
change in the protein’s apparent melting
temperature (Tm), which can be measured
using DSC. An increase in Tm upon binding
typically correlates with better Ki values
and thus increased ligand affinity (Celej
et al., 2006; Hellman et al., 2016). A com-
plex of Mtb DHFR and NADPH was used
as the control sample to provide a compar-
ison between the Tm of the uninhibited and
inhibited protein. Overall, compound bind-
binding is rapidly or slowly reversible. In brief, Mtb DHFR was ing resulted in an increase in Tm (Table 1). UCP1172 and MTX
incubated with a stoichiometric equivalent of inhibitor at concen- showed the largest stabilizing effects on the protein (81ꢁC and
trations above Ki for 3 h to allow the formation of the EI complex. 75.5ꢁC, respectively) compared with the control (65ꢁC).
The assay mixture was then diluted 100-fold into assay buffer UCP1175 and UCP1063 also showed an increased thermal sta-
containing a saturating concentration of DHF to disturb the equi- bility (72.9ꢁC and 71ꢁC, respectively), although to a lesser extent
librium. The rapid dilution favors ligand dissociation while disfa- compared with UCP1172 and MTX. Binding of PAS-M caused
voring ligand rebinding. The recovery of enzymatic activity was the smallest shift in Tm, again suggesting its weak affinity for
followed as a function of time after dilution. Under the condition Mtb DHFR. In sum, the changes in thermal stability of Mtb
of minimal inhibitor rebinding, the observed rate constant, kobs
can be considered as a reasonable estimate of the dissociation
rate constant koff. The off-rate constant was then used to esti- Structural Studies of Mtb DHFR in Complex with INCAs
mate the target-inhibitor residence time (tR) (Table 1).
and PAS-M
,
DHFR upon binding correlates well with Ki values.
After dilution, enzymatic activity was recovered for all com- To investigate the ligand-target interactions that drive affinity
pounds over the assay time frame (Figure S1). The resulting and potency, we co-crystallized UCP1172, UCP1175, and
progress curves for INCAs, UCP1063, and MTX were curvi- PAS-M with Mtb DHFR and solved the protein ternary complex
linear (a lag phase followed by a linear phase), suggesting bound to the inhibitors and NADPH. UCP1172 and UCP1175
slowly reversible behavior. UCP1172 showed the longest resi- bind in a similar mode to that reported for MTX (Li et al., 2000).
dence time on Mtb DHFR (42 min) and, as a result, the enzy- The 2,4-diaminopyrimidine ring of INCAs is deeply positioned
matic activity recovery in the presence of UCP1172 was slower into the hydrophobic pocket making two hydrogen bonds with
than the other compounds. The residence time of UCP1175 the side chain of Asp27 and two hydrogen bonds with the
and UCP1063 were almost similar (14 and 10 min, respec- main chain of Ile5 and Ile94 (Figure 3, top left and right). The hy-
˚
tively). The progress curve for PAS-M was linear with a slope droxyl of Tyr100 is also 3.4 A away from the 4-amino group. Wa-
nearly equal to the slope of the uninhibited control sample, sug- ter-mediated interactions between the 2,4-diaminopyrimidine
gesting fast dissociation. The tR for PAS-M was determined to ring and Trp22, His30, and Ile113 and p-p stacking interactions
be 1.5 min. Overall, a correlation between Ki and tR was with Phe31 are present. The biaryl system of INCAs makes hy-
observed, indicating that the dissociation rate is influenced by drophobic interactions with Gln28, Leu50, Pro51, Ile54, and
the affinity of the inhibitor for the target. The data suggest Leu57. The propargylic methyl of UCP1172 (Figure 3, top left) im-
that unlike MTX and INCAs, PAS-M has a weak affinity for proves its hydrophobic interactions compared with UCP1175,
Mtb DHFR. However, given the nature of PAS’s bioactivation, which lacks propargylic functionality. The carboxylate of INCAs
in vitro enzyme assay is a poor metric for PAS-M’s Mtb forms a hydrogen bond with a highly conserved Arg60, which
DHFR activity in the cell. PAS-M biosynthesis requires a stoi- is likely the interaction that defines the increased affinity of
chiometric reduction in DHF, thereby reducing the natural sub- INCAs. Although our efforts to co-crystallize UCP1063 with
strate that PAS-M competes with for Mtb DHFR. Therefore, a Ki Mtb DHFR were not successful, we believe the replacement of
value measured in an in vitro assay may drastically underesti- benzoic acid with pyridine in UCP1063 is responsible for its
mate PAS’s effect on the organism.
decreased affinity for the enzyme compared with UCP1172.
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Table 1. Microbiological and Biochemical Characteristics of INCAs, PAS-M, and MTX
Mtb DHFR
Ki (nM)a
Compounds
MTX
MIC (mg/mL)
koff (minꢀ1 b
)
tR (min)c
18 1.5
42 5.3
14 1.5
10.5 1.6
1.5 0.3
ND
Tm (ꢁC)d
76 3.5
Hu DHFR Ki (nM)
0.7 0.11
Mtb Rv2671 Ki (nM)
ND
>100
0.03
0.125
2
1.6 0.16
0.91 0.15
6.55 0.26
0.056 0.006
0.024 0.003
0.072 0.008
0.096 0.015
0.643 0.125
ND
UCP1172
UCP1175
UCP1063
PAS-M
81
3
60 15
80 6.5
73 3.2
71 2.7
68 1.8
ND
51
3
1,170 140
52 6.2
8
1.3
11.1 1.3
ND
0.5e
750 120
9.1 0.25
9.9 0.62
27,500 3500
350 12.5
120 8.5
UCP1163
4
120 7.5
144 3.5
UCP1164
0.5
ND
ND
ND
aCalculated as Ki = IC50/(1+[S]/Km).
bkoff is assumed to be equal to the activity recovery rate constant from jump-dilution assay.
cCalculated as the reciprocal of the rate constant (kobs).
dThe Tm of the control (Mtb DHFR + NADPH) was determined to be 65ꢁC.
eThis value is the MIC of PAS. The MIC of PAS-M was determined to be 8 mg/mL (the difference is most likely due to permeability issues). All the
experiments are performed in triplicates.
The co-crystal structure of Mtb DHFR bound to PAS-M has re- In Hu DHFR, the corresponding Arg70 is too far from the carbox-
˚
vealed a likely reason for its poor affinity. Compared with MTX ylate of UCP1172 to form a hydrogen bond (6.0 A). Although the
˚
(Li et al., 2000), the pteridine ring of PAS-M is in an inverse orien- carboxylate is near Asn64 (3.2–3.6 A), the angle does not seem to
tation, with N1 and N8 too far for direct or water-mediated favor hydrogen bonding. The lack of optimized interactions be-
hydrogen bonds (Figure 3, middle left). The N1 and 2-amino tween the carboxylate group of UCP1172 and the Hu DHFR
group of the pteridine ring retain two hydrogen bonds with active site residues is likely the reason for its weaker affinity for
Asp27. The pteridine carbonyl and N5 make water-mediated in- Hu DHFR compared with Mtb DHFR (Table 1).
teractions with Trp22, His30, and Thr113. The glutamate tail
forms hydrogen bonds with Arg60 and Arg32. A large water Inhibition of Mtb Rv2671
network was also observed between the hydroxyl group and To further evaluate the effect of INCAs and PAS-M on Mtb
the residues of the binding pocket. Despite these interactions, folate metabolism, we tested the potency of INCAs and
the inability of the pteridine ring to interact with the highly PAS-M against Mtb RV2671, an enzyme in Mtb recently shown
conserved Ile5, Ile94, and Tyr100 is likely responsible for PAS- to possess DHFR activity (Cheng and Sacchettini, 2016). Over-
M’s weak affinity for Mtb DHFR. The flipped pteridine ring has expression of Rv2671 has been shown to be associated with
also been observed in co-crystal structures of human DHFR resistance to PAS and antifolates (Zhang et al., 2015; Zheng
bound to folate and is likely responsible for the difference in af- et al., 2013).
finity between folate and MTX for Hu DHFR (Oefner et al., 1988).
The enzyme activity of Mtb Rv2671 was measured by
following NADPH oxidation at 340 nm, and its kinetic parameters
were determined (Table S1). The Km of Mtb Rv2671 for DHF was
Selectivity of INCAs toward the Microbial DHFR
Previously, we have shown that several INCAs, including determined to be 39 mM, about four times higher than the Km for
UCP1172, have no measurable cytotoxicity toward mammalian Mtb DHFR. The kcat of Mtb Rv2671 is 0.69 sꢀ1, which is almost
cells at concentration up to 100 mM (Scocchera et al., 2016). 2.5 times less than Mtb DHFR. These numbers agree with
The low toxicity of moderately potent DHFR inhibitors such as what is known, that Mtb Rv2671 is less efficient as a reductase
the INCAs, may be attributed to the unique way in which human than DHFR (Cheng and Sacchettini, 2016).
DHFR is regulated. In humans, DHFR is known to bind its
We then evaluated the inhibitory activity of INCAs and PAS-M
cognate RNA, and the presence of a DHFR inhibitor leads to against Mtb Rv2671 (Table 1). Compounds UCP1172 and
dissociation, relieving translational arrest, and rapid translation UCP1063 showed potent inhibition of Mtb Rv2671, with Ki values
of DHFR. This provides a rapid, intrinsic protection to human of 82 and 53 nM, respectively. UCP1175 showed moderate inhi-
cells that is unavailable to prokaryotic pathogens (Zhang and Ra- bition of the enzyme with a Ki of 1,170 nM. PAS-M did not
thod, 2002). Here, to understand the affinity differences between exhibit a significant inhibitory effect against Mtb Rv2671 (Ki =
Mtb DHFR and Hu DHFR we co-crystallized Hu DHFR with 27,500 nM). To assess the impact of Rv2671 inhibition on the
UCP1172 (Figure 3, middle right). The interactions of the 2,4-di- antimycobacterial activity of the compounds, we used an enan-
aminopyrimidine ring that anchors UCP1172 in the active site are tiomerically pure INCA couple, UCP1164 and UCP1163 (Fig-
very similar to what was observed in the Mtb DHFR and ure 2), that possess similar DHFR inhibitory activity but different
UCP1172 complex. The 4-amino moiety forms a hydrogen inhibition against Rv2671. UCP1164 and UCP1163 Ki values
˚
bond with the backbone carbonyl of Ile7 (3.0 A) and another against Rv2671 were determined to be 120 and 350 nM, respec-
hydrogen bond with the backbone carbonyl oxygen of Val115 tively (Table 1).
˚
(3.3 A). Atom N1 and the 2-amino group form two strong
Interestingly, when tested against the organism, UCP1164
hydrogen bonds with Glu30 (2.8 and 2.6 A, respectively). Glu30 showed more potent inhibition of cell growth with a minimum
corresponds to the conserved Asp27 among bacterial DHFRs. inhibitory concentration (MIC) value of 0.5 mg/mL compared
˚
4
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Figure 3. Structural Analysis of Mtb DHFR, Hu DHFR, and Rv2671
Top left: Mtb DHFR in complex with NADPH (dark gray) and UCP1172 (light gray) at 1.4 A (PDB: 6DDP). Top right: Mtb DHFR in complex with NADPH and
˚
˚
UCP1175 (yellow) at 1.7 A (PDB: 6DDS). Middle left: Mtb DHFR in complex with NADPH and PAS-M (brown) at 1.4 A (PDB: 6DDW). Middle right: Hu DHFR in
˚
˚
complex with NADPH and UCP1172 (light gray) at 2.4 A (PDB: 6DE4). Bottom: Mtb Rv2671 in complex with NADPH and UCP1063 (cyan).
with UCP1163 (MIC = 4 mg/mL) (Table 1). Due to high structural anti-parallel b strand, seven a helices, and one 310 helix (h1).
similarity between these two compounds, we do not expect As reported previously (Cheng and Sacchettini, 2016), a weak
them to have significant permeability differences. Therefore, density for the nicotinamide ribose moiety of NADPH and resi-
the observed difference in MIC values is most likely caused by dues 90–95 was observed. This disorder is likely due to the lack
different enzyme inhibitory activity against Mtb Rv2671. Taken of stabilized interactions in these regions and explains the high
together, the data suggest that dual inhibition of Mtb DHFR Km for DHF and NADPH. The 2,4-diaminopyrimidine ring of
and Rv2671 contributes to potent antimycobacterial activity INCAs makes direct hydrogen bonds with Asn44, Asp67
of INCAs.
(corresponding to Asp27 in Mtb DHFR), and Glu193. In addi-
tion, there is a water-mediated interaction with Thr214 and
p-p stacking with Phe71 (corresponding to Phe31 in Mtb
Structural Studies of Mtb Rv2671 Bound to UCP1063
To investigate drug-target interactions, We co-crystallized Mtb DHFR). The pyridine nitrogen of UCP1063 is too far from
Rv2671 in complex with NADPH and UCP1063 (Figure 3, bot- Asn72 and Arg68 to form direct hydrogen bonds (5.2 and
˚
tom). The protein is composed of eight parallel b strands, one 4 A, respectively).
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FDTS reaction is the reduced folate THF, not DHF. Approxi-
mately 5% of microbial species, including Mtb, express all three
Table 2. Genetic Determinants of PAS Resistance in Mtb
Spontaneous Mutants and their Susceptibility to PAS and INCAs
folA, thyA, and thyX. Although it is unclear why both thyA and
thyX genes are maintained in such organisms, studies have
shown that thyX is essential for Mtb growth and survival, and
its overexpression is associated with PAS resistance in clinical
isolates (Fivian-Hughes et al., 2012; Mathys et al., 2009; Moradi-
garavand et al., 2016). Considering poor PAS-M affinity for Mtb
DHFR and Rv2671, and PAS-M resistance due to thyX overex-
pression, we tested the inhibitory potential of PAS-M against
Mtb FDTS. Due to the structural similarities between PAS-M
and mTHF its ability to bind FDTS is likely.
(MIC in mg/mL)
Mutation
folC
folC
(I43T)
32
thyX
WT Mtb
(E153G)
32
(-16 C- > T)
PAS
0.5
16
1
UCP1172
UCP1175
0.03
0.125
0.125
4
0.015
1
16
In Vitro Selection of PAS-Resistant Mtb Mutants
We performed DSC experiments to determine PAS-M binding
The study of PAS-resistant clinical isolates and spontaneous to Mtb FDTS via its effect on protein stability. A mixture of FDTS,
mutants has revealed no mutations in the dfrA gene; instead, dUMP, and FAD was used as the control. DSC showed the a Tm
mutations were identified in thyA, folC, thyX, and Rv2671 (Minato of 66.6ꢁC for the control sample and 71.9ꢁC for the sample incu-
et al., 2015; Moradigaravand et al., 2016; Zhang et al., 2015). This bated with PAS-M (Figure 4, top left). This increase in Tm sug-
seems to suggest other mechanisms of actions for PAS-M rather gests the stabilizing effect of PAS-M on Mtb FDTS due to
than DHFR inhibition. To improve our understanding of the un- binding.
derlying mechanisms of action of PAS, and its resistance, we
It is known that mTHF and NADPH binding sites in FDTS
performed in vitro generation of PAS-resistant isolates and partially overlap, and that high concentrations of mTHF inhibit
investigated the mutations associated with PAS resistance NADPH oxidation due to competition for the binding site (Hunter
(Table 2). Genome sequencing revealed folC (encoding for dihy- et al., 2008; Koehn et al., 2012). A ligand that competes for the
drofolate synthase, DHFS) mutations (E153G and I43T) in two of mTHF binding site should interrupt NADPH binding and ulti-
the mutants. Both mutations have been previously shown to mately oxidation event. The inhibitory effect of PAS-M, MTX,
confer resistance to PAS in clinical isolates of Mtb (Zhao et al., and INCAs on the NADPH oxidation activity of Mtb FDTS was
2014). It has been suggested PAS converts to its metabolite by tested by monitoring absorbance at 340 nM (Figure 4, top right).
acting as a competing substrate for dihydropteroate synthase All compounds were tested at stoichiometric equivalents of
(DHPS) (encoded by folP1) and DHFS, sequentially (Chakraborty NADPH (200 mM). mTHF was used as the positive control with
et al., 2013). Residues E153 and I43 in DHFS are important for 100% inhibition of NADPH oxidation. Excitingly, PAS-M showed
dihydropteroate (H2Pte) binding. These mutants were shown to an inhibitory effect similar to mTHF, fully inhibiting NADPH oxida-
be much less efficient at catalyzing the glutamination of both tion. At the same concentration, MTX showed ꢂ50% inhibition,
the natural substrate (H2Pte), and the PAS derivative (hydroxyl- and no significant inhibition was observed with INCAs (less
H2Pte). A mutation here would lead to a reduction in intracellular than 5%). We performed a dose-response NADPH oxidation in-
[PAS-M], and the organism would be spared of its antibacterial hibition assay to estimate the IC50 of PAS-M for the NADPH
effects. We also identified a PAS-resistant isolate with a C to T oxidation reaction (Figure S2). The IC50 was determined to be
upstream mutation at position ꢀ16 that has been previously 14.4 1.5 mM.
found to result in thyX overexpression and PAS resistance (Mor-
Although the NADPH oxidation assay is considered a valid
adigaravand et al., 2016; Zhang et al., 2013, 2015). The PAS- method to identify FDTS inhibitors, it is not a direct measurement
resistant isolates remained susceptible to INCAs, although at of dTMP synthesis. To evaluate the inhibitory effect of PAS-M on
slightly higher MIC values (especially for UCP1175). The largest the production of dTMP, we monitored the formation of dTMP in
increase in the MIC of INCAs was observed against the thyX FDTS reaction at 25ꢁC by employing high-pressure liquid chro-
overexpressor strain. The fact that thyX overexpression is a matography. The uninhibited reaction was used as the control,
PAS resistance mechanism suggests that either the organism and samples were taken at various times (0, 10, 20, and
is compensating for DHFR inhibition by PAS-M, or that PAS/ 30 min). Compared to the control, there is a significant decrease
PAS-M directly interacts with FDTS.
(ꢂ2.5-fold) in dTMP levels in the reaction incubated with PAS-M
at stoichiometric equivalents with NADPH (100 mM) (Figure 4,
middle left and right). These data suggest that PAS-M inhibits
Inhibition of FDTS
In human cells and almost 70% of microbial species, thymidylate the formation of dTMP by the Mtb FDTS. To the best of our
synthase (TS) (encoded by thyA) catalyzes the reductive methyl- knowledge, this is the first report of PAS-M’s inhibitory effect
ation of deoxyuridine monophosphate (dUMP) using methylene- on the Mtb FDTS.
THF (mTHF) as both a one-carbon donor, and a reductant, to
generate dTMP, an essential building block of DNA. However, Modeling PAS-M into FDTS
in approximately 30% of microbial species, de novo synthesis Our efforts to co-crystallize PAS-M with Mtb FDTS were not suc-
of dTMP relies on FDTS (encoded by thyX). FDTS uses NADPH cessful, even when attempting to soak PAS-M into an
as the source of hydride, mTHF as the one-carbon donor and FDTS:FAD:dUMP complex. However, we solved a Mtb FDTS
˚
FAD as a relay system for hydride and methylene transfer, to structure bound to FAD and dUMP at 3.5 A resolution. We
catalyze the formation of thymidylate (Mishanina et al., 2016; used this crystal structure for our structural analysis and
Myllykallio et al., 2003). Unlike TS, the folate product of the modeled PAS-M into the Mtb FDTS binding site. Mtb FDTS is
6
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Figure 4. Assessment of FDTS Inhibition by PAS-M Compound
Top left: differential scanning calorimetry analysis of PAS-M binding to Mtb FDTS. Top right: effect of INCAs, MTX, and PAS-M on the NADPH oxidation activity of
Mtb FDTS. Data are presented as means SD, in three independent experiments. Middle left: high-pressure liquid chromatography (HPLC) analysis of Mtb FDTS
reaction (uninhibited). Middle right: HPLC analysis of Mtb FDTS reaction inhibited by PAS-M. Bottom: modeling of PAS-M (brown) into the binding pocket of Mtb
FDTS in complex with FAD (yellow) and dUMP (magenta).
a tetramer with each monomer composed of a central a/b highly conserved among different species, and its mutation
domain flanked by two helical top and bottom domains (Fig- has been shown to decrease the catalytic activity of FDTS
ure S3). The four monomers (a, b, c, and d) come together to from Thermotoga maritima (Koehn et al., 2012). The 2-amino
˚
form a tetrameric molecule with four active sites. The adenine group of the pteridine ring is 3.0 A from Tyr44 of subunit c. The
˚
ring of FAD is buried in a deep binding pocket in the enzyme. characteristic hydroxyl group of PAS-M is 3.1 A from the
˚
The adenosine monophosphate and flavin mononucleotide conserved Arg190 and 3.5 A from the phosphate groups of the
(FMN) moieties of FAD interact with conserved histidine and argi- FMN moiety of FAD. The carboxylates in the glutamate tail of
nine residues in the binding pocket. The isoalloxazine ring forms PAS-M are within hydrogen bonding distance from Arg193,
hydrogen bonds with Arg95 of subunit a, Gln103 of subunit d, Arg190, and His194. We also assume that water-mediated inter-
and Tyr144 of subunit c, and stacks against the pyrimidine ring actions would further improve PAS-M affinity for Mtb FDTS.
of dUMP. We then modeled PAS-M into the Mtb FDTS:FAD:
¨
dUMP complex using molecular modeling software (Schro- Mutation Rate and Emergence of Resistance to INCAs
dinger). In this model, the pterin moiety of PAS-M is stacked be- To evaluate the development of resistance to INCAs and esti-
tween the isoalloxazine ring of FAD (which itself is stacked mate mutation rates in vitro we performed fluctuation analysis
against the pyrimidine ring of dUMP) and the imidazole of to select spontaneously resistant mutants. Mutations conferring
His69 of subunit c (Figure 4, bottom). This histidine residue is resistance to INCAs were measured to occur at the rate of
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Figure 5. The Effects of UCP1172 and PAS on Mtb Mycolic Acid Profile
Left: autoradiograph of TLC analysis of lipids extracted from 14C acetate-labeled Mtb Erdman cells treated with UPC1172, PAS, or DMSO as a control (CTR);
autoradiograph of standard (middle) and argentation (right) TLC analysis of methyl esters of fatty and mycolic acids isolated from 14C acetate-labeled Mtb Erdman
cells treated with UPC1172, PAS, or DMSO as a control (CTR).
2.4 3 10ꢀ8 and 8.5 3 10ꢀ8 for UCP1172 and UCP1175, pholipids, trehalose monomycolates, or trehalose dimycolates
respectively (Table S2). This is similar to the rate reported for (Figure 5, left). However, total incorporation of 14C into the frac-
isoniazid, streptomycin, and bedaquiline (Andries et al., 2005; tions of extracted lipids was reduced compared with the non-
Huitric et al., 2010). The MIC values of the INAC-R strains treated control (83% and 66% by 0.72 and 1.44 mg/mL
were elevated by 32-fold; however, owing to the high potency UCP1172, respectively, and 82% and 69% by 0.36 and
of UCP1172 against the wild-type organism, the MIC of resis- 0.72 mg/mL PAS, respectively). Analysis of fatty acid and mycolic
tant strains only increases to the level of 1 mg/mL. The mutation acid methyl esters (FAME/MAME) extracted from radiolabeled
rate reported for PAS is 2 3 10ꢀ7, with MIC values increasing cells revealed important changes in both UCP1172- and PAS-
up to 8 mg/mL(Zhao et al., 2014). The mutation rate of INCAs treated mycobacteria. An overall reduction of the 14C incorpora-
against methicillin-resistant Staphylococcus aureus (MRSA) is tion to the FAME/MAME fraction was observed (14%, 36%, and
lower than what we observe for Mtb (Reeve et al., 2016). This 56% reduction by 0.36, 0.72, and 1.44 mg/mL UCP1172, respec-
is most likely due to lack of compensatory enzymes such as tively, and 52% reduction by 0.36 and 0.72 mg/mL PAS). Interest-
Rv2671 and FDTS in MRSA, an important distinction between ingly, both PAS and UCP1172 treatment led to a significant
Mtb and other common bacterial pathogens. Interestingly, reduction of methoxymycolic acids (Figure 5, middle). For both
resistance to INCAs also confers cross-resistance to PAS, PAS and UCP1172, further analysis of mycolic acids by argenta-
showing a similar 32-fold loss of potency but with an MIC of tion thin-layer chromatography (TLC) revealed the appearance of
16 mg/mL. This also provides additional evidence that the major other forms of mycolates, which were not present in non-treated
mechanism of antimycobacterial action of the INCAs is through Mtb (Figure 5, right). Slower migration of these forms indicates
blockade of the folate pathway.
the presence of mono- or di-unsaturation in their carbon chains.
Since these forms of mycolic acids are precursors for further
modifications with SAM-dependent methyltransferases, we
conclude that both UCP1172 and PAS interfere with these
The Inhibitory Effect of PAS and UCP1172 on Mycolic
Acid Production
It has been shown that antifolate treatment in Mtb leads to a crit- methylation processes. It has been shown that disruption of
ical disruption of the activated methyl cycle and decreases pools the folate pathway leads to perturbation of the activated methyl
of methionine and S-adenosylmethionine (SAM) (Nixon et al., cycle and altered gene regulation involved in the biosynthesis
2014). SAM is responsible for the supply of reactive methyl and use of methionine and SAM, including methyltransferases
groups for a myriad of important cellular processes, including dependent on SAM (Nixon et al., 2014). We thus conclude that
the formation of a variety of functional groups in the meromycolic the observed effects (decrease in all forms of standard myco-
chain of mycolic acids that define several mycolate types in my- lates and accumulation of unsaturated mycolates) is most likely
cobacteria (Boissier et al., 2006). Given the link between the due to a lack of SAM for SAM-dependent methyltransferases
folate pathway and the activated methyl cycle, we hypothesized involved in the modifications of mycolic acids.
that treatment with INCAs or PAS may lead to changes in the lipid
composition of the Mtb cell wall, particularly related to mycolic DISCUSSION
acids. We performed 14C acetate labeling of model strain Mtb
Erdman grown in the presence of UCP1172, which was added Functional redundancy is commonly observed in Mtb and poses
in early mid-log phase of growth at 0.36, 0.72, and 1.44 mg/mL a challenge for drug discovery against this resilient pathogen
final concentrations. As a control, the bacteria were treated (Barkan et al., 2010; Ganapathy et al., 2015; Johnston et al.,
with PAS at 0.36 and 0.72 mg/mL final concentrations in the 2010; van der Veen and Tang, 2015). One way to address this
same conditions. The treatment with UCP1172 or PAS did not problem is multiple targeting of these functional analogs by
lead to any dramatic alterations in the synthesis of major phos- use of a single pharmacophore. In fact, many successful
8
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monotherapeutic agents with low endogenous resistance 1 mg/mL, suggesting it may be possible to suppress these mu-
rate have multiple targets (Silver, 2007, 2011). Although TB tants at an achievable in vivo concentration.
monotherapy is likely an unrealistic goal, anti-TB agents with
multiple targets can simplify the regimen and improve treatment sues associated with current drug regimens and to improve TB
outcomes. therapy outcomes. Our work on targeting the folate pathway in
New anti-TB agents and targets are required to address the is-
In this report, we have shown that both INCAs and PAS have TB highlights the unique nature of this pathway relative to other
multiple targets within the Mtb folate pathway. Biochemical pathogenic bacterial, and suggests that it is possible to achieve
studies indicate that INCA antimycobacterial activity is due to highly potent agents by targeting multiple enzymes along the
potent and long-lasting inhibition of canonical and non-canoni- Mtb folate pathway. Downstream effects of the interrupted folate
cal DHFR enzymes in Mtb. The importance of dual inhibition of pathway in Mtb are manifold and include changes in many
Mtb DHFR and Mtb Rv2671, along with slow dissociation of essential cell functions such as DNA and protein biosynthesis
the ligand from Mtb DHFR in improving the antimycobacterial and cell wall metabolism. Continued efforts will have to consider
activity of INCAs is reflected in the excellent MIC value of the enzymes of the alternate pathway when designing antifolates
UCP1172 against Mtb (MIC = 0.03 mg/mL) (Table 1).
as effective anti-TB agents.
The elegant work by Chakraborty et al. (2013) and Zheng et al.
(2013) showed that PAS is a competing substrate for DHPS and
acts as a prodrug for DHFR. Our data elucidate the kinetics of
PAS-M action against DHFR and reveals FDTS as a likely second
target. To the best of our knowledge, inhibition of FDTS as part of
the mechanism of action of PAS has not been reported before.
This suggests that the downstream effects of PAS mis-incorpo-
ration into the folate pathway is more complicated than previ-
ously thought. We propose that PAS exerts its antimycobacterial
activity by a multi-targeted effect on the folate metabolism rather
than acting on a single target within the pathway.
SIGNIFICANCE
Tuberculosis therapy can specifically benefit from multi-tar-
geting agents because Mycobacterium tuberculosis, the
pathogen responsible for human tuberculosis, is treated by
a long-term combination of drugs. We show that com-
pounds with multiple targets within the M. tuberculosis
folate pathway effectively inhibit the growth of live bacteria
by disruption of essential cellular functions including DNA
biosynthesis and cell wall metabolism. We also show that
para-aminosalicylic acid (PAS), a TB drug used for years,
has multiple targets and thus has provided a good example
of a successful multi-targeted agent against tuberculosis.
Finally, we have shown that antifolate treatment in Mtb results
in a dramatic decrease in the level of all forms of standard my-
colic acids, essential components of the mycobacterial cell
wall. This adds to the previous report by Nixon et al. (2014),
which indicated that antifolate treatment in Mtb disrupts the acti-
vated methyl cycle. They showed that treatment of Mtb cells with
WR99210, a DHFR inhibitor, caused a 20-fold decrease in the
concentration of SAM. SAM serves as the donor of reactive
methyl groups for methylation of DNA, RNA, proteins, and lipids
in reactions catalyzed by SAM-dependent methyltransferases.
In Mtb, at least six SAM-dependent methyltransferases (en-
coded by mmaA1–mmaA4, cmaA2, and pcaA genes) were iden-
tified to catalyze the formation of mycolic acid chain (Barkan
et al., 2009). These studies support the notion that inhibiting
the production of reduced folates interrupts the biosynthesis of
mycolic acids, most likely due to the interruption of the activated
methyl cycle.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
d CONTACT FOR REAGENTS AND RESOURCE SHARING
B Bacterial Strains and Culture Conditions
d METHOD DETAILS
B Synthesis of PAS-M and Compound Characterization
B In Vitro Susceptibility Testing
B Transformation, Expression and Purification of Mtb
DHFR, Hu DHFR, Mtb Rv2671, and Mtb FDTS
B Enzyme Inhibition Assay for Mtb DHFR, Hu DHFR, and
Mtb Rv2671
In the Mtb folate pathway, functional redundancy is used to
ensure high levels of THF, considering that DHFR, Rv2671, and
FDTS all produce THF. We have identified compounds that
target different pairs of these enzymes, but it is too enticing to
not consider a compound capable of targeting all three enzymes.
Such a compound would shut down all viable THF-generating
enzymes and should prove highly potent against the organism.
The work presented here can be further used in structure-based
design of such inhibitors. It is important to note that the rate of
mutation to INCAs suggests that compounds that inhibit multiple
targets within the same pathway (series inhibition) are still prone
to resistance as a single mutation can relieve inhibition of the sole
essential pathway being targeted. This effect differs from clas-
sical combination chemotherapy approaches where multiple
essential pathways are being simultaneously inhibited. Excit-
ingly, the high initial potency of INCAs (UCP1172), means
that the elevated MIC in the INCA-R strains only increases to
B Jump-Dilution Recovery Assay
B Mtb FDTS NADPH Oxidation Assay
B Crystallization of Mtb DHFR, Data Collection, and
Structure Determination
B Crystallization of Hu DHFR, Data Collection, and Struc-
ture Determination
B Crystallization of Mtb Rv2671, Data Collection, and
Structure Determination
B Crystallization of Mtb FDTS, Data Collection, and
Structure Determination
B Selection of PAS-Resistant Mutants
B Determination of Mutation Rates
B DNA Extraction from PAS-Resistant Isolates
B Whole Genome Sequencing
B Modelling of PAS-M into Mtb FDTS
Cell Chemical Biology 26, 1–11, June 20, 2019
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´
Celej, M.S., Dassie, S.A., Gonzalez, M., Bianconi, M.L., and Fidelio, G.D.
B Analysis of Lipids and Mycolic Acids
B Differential Scanning Calorimetry (DSC)
B HPLC Analysis of Mtb FDTS Reaction
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND SOFTWARE AVAILABILITY
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(2013). para-Aminosalicylic acid acts as an alternative substrate of folate meta-
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SUPPLEMENTAL INFORMATION
Supplemental Information can be found online at https://doi.org/10.1016/j.
chembiol.2019.02.013.
Copeland, R.A. (2010). The dynamics of drug-target interactions: drug-target
residence time and its impact on efficacy and safety. Expert Opin. Drug
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ACKNOWLEDGMENTS
Copeland, R.A., Basavapathruni, A., Moyer, M., and Scott, M.P. (2011). Impact
of enzyme concentration and residence time on apparent activity recovery in
jump dilution analysis. Anal. Biochem. 416, 206–210.
We acknowledge that this research was funded by the US NIH (AI104841 and
AI111957). We thank Stanford Synchrotron Radiation Lightsource (SSRL) and
National Synchrotron Light Source II (NSLS-II) at Brookhaven National Lab for
the beamline access and their staff members for their assistance. We also like
to thank University of Connecticut Center for Open Research Sources and
Equipment (COR2E) and Microbial Analysis, Resources, and Services
(MARS). K.M. and J.K. also acknowledge support by the Ministry of Education,
Science, Research and Sport of the Slovak Republic (grant VEGA 1/0301/18
and 0395/2016 for Slovak/Russian cooperation in science 2015–15075/
33841:1-15E0).
Dawadi, S., Kordus, S.L., Baughn, A.D., and Aldrich, C.C. (2017). Synthesis
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AUTHOR CONTRIBUTIONS
Ford, C.B., Lin, P.L., Chase, M.R., Shah, R.R., Iartchouk, O., Galagan, J.,
Mohaideen, N., Ioerger, T.R., Sacchettini, J.C., Lipsitch, M., et al. (2011).
Use of whole genome sequencing to estimate the mutation rate of
Mycobacterium tuberculosis during latent infection. Nat. Genet. 43, 482–486.
Conceptualization and methodology, B.H., E.S., and D.W.; Investigation, B.H.,
´
´
E.S., C.S., J.Krucinska, K.V., N.G.-D., H.E., A.E., K.M., and J.Kordulakova.;
Writing, B.H., E.S., and D.W.; Visualization, B.H. and E.S.; Funding Acquisition,
D.W. and K.M.; Supervision, M.C. and D.W.
Ford, C.B., Shah, R.R., Maeda, M.K., Gagneux, S., Murray, M.B., Cohen, T.,
Johnston, J.C., Gardy, J., Lipsitch, M., and Fortune, S.M. (2013).
Mycobacterium tuberculosis mutation rate estimates from different lineages
predict substantial differences in the emergence of drug-resistant tubercu-
losis. Nat. Genet. 45, 784–790.
DECLARATION OF INTEREST
D.W. is a co-founder of Quercus Molecular Design.
Ganapathy, U., Marrero, J., Calhoun, S., Eoh, H., de Carvalho, L.P.S., Rhee, K.,
and Ehrt, S. (2015). Two enzymes with redundant fructose bisphosphatase ac-
tivity sustain gluconeogenesis and virulence in Mycobacterium tuberculosis.
Nat. Commun. 6, 7912.
Received: August 24, 2018
Revised: January 22, 2019
Accepted: February 24, 2019
Published: March 28, 2019
Hajian, B., Scocchera, E., Keshipeddy, S., G-Dayanandan, N., Shoen, C.,
Krucinska, J., Reeve, S., Cynamon, M., Anderson, A.C., and Wright, D.L.
(2016). Propargyl-linked antifolates are potent inhibitors of drug-sensitive
and drug-resistant Mycobacterium tuberculosis. PLoS One 11, e0161740.
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STAR+METHODS
KEY RESOURCES TABLE
REAGENTS OR RESOURCES
SOURCE
IDENTIFIER
Chemical, Peptides, and Recombinant Proteins
Beta-nicotinamide adenine dinucleotide phosphate (NADPH)
Dihydrofolic acid (DHF)
Alfa-Aesar
Cat#J60387
Cat#D7006
Cat#T3125
Cat#A6770
Cat#I3377
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Sigma-Aldrich
Alfa-Aesar
Tetrahydrofolic acid (THF)
Methotrexate
Isoniazid
Flavin adenine dinucleotide disodium salt hydrate (FAD)
2’-deoxyuridine-5’-monophosphate disodium salt (dUMP)
Thymidine 5’-monophosphate disodium salt (dTMP)
5,10-Methylenetetrahydrofolate (mTHF)
Isopropyl-1-thio-b-D-galacto-pyranoside (IPTG)
para-Aminosalicylic acid
Cat#F6625
Cat#J64627
Cat#T7004
Cat#F680350
Cat#R0392
Cat#A79604
Cat#FC-131
Cat#A63880
Sigma-Aldrich
Toronto Research Chemicals
Thermo Scientific
Sigma-Aldrich
Illumina
Nextera XT DNA library preparation kit
AMPure XP beads
Beckman-Coulter
Bacterial and Virus Strains
Mycobacterium tuberculosis Erdman Strain
BL21 (DE3) competent E. Coli
ATCC
Cat#35801
Cat#C2527I
New England BioLabs
Recombinant DNA
Plasmid pET-28a(+)-MtbRv271
GenScript
GenScript
GenScript
GenScript
N/A
N/A
N/A
N/A
Plasmid pET-24d-MtbFDTS
Plasmid pET-41a(+)-MtbDHFR
Plasmid pET-41a(+)-HuDHFR
Deposited Data
Structure of Mtb DHFR bound to UCP1172
Structure of Mtb DHFR bound to UCP1175
Structure of Mtb DHFR bound to PAS-M
Structure of Hu DHFR bound to UCP1172
Structure of Mtb Rv2671 bound to UCP1063
Whole Genome Sequence of Wild-type Mtb
Whole Genome Sequence of PAS-R folC (E153G) mutant
Whole Genome Sequence of PAS-R folC (I43T) mutant
Whole Genome Sequence of PAS-R thyX mutant
This study
This study
This study
This study
This study
This paper
This paper
This paper
This paper
PDB ID: 6DDP
PDB ID: 6DDS
PDB ID: 6DDW
PDB ID: 6DE4
PDB ID: 6DE5
BioSample accession: SAMN10962817
BioSample accession: SAMN10962818
BioSample accession: SAMN10962820
BioSample accession: SAMN10962819
CONTACT FOR REAGENTS AND RESOURCE SHARING
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact,
Dennis L. Wright (dennis.wright@uconn.edu).
Bacterial Strains and Culture Conditions
Mtb Erdman (ATCC 35801) was purchased from the American Type Culture Collection (Manassas, VA). The organism was grown in
modified 7H10 Broth (pH 6.6; 7H10 agar formulation with agar and malachite green omitted) supplemented with 10% Middlebrook
oleic acid-albumin-dextrose-catalase (OADC) enrichment and 0.05% Tween 80 on a rotary shaker at 37ꢁC for 7-10 days.
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Please cite this article in press as: Hajian et al., Drugging the Folate Pathway in Mycobacterium tuberculosis: The Role of Multi-targeting Agents, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.013
METHOD DETAILS
Synthesis of PAS-M and Compound Characterization
The synthesis of PAS-M was derived from a recent report by Dawadi et al. (Dawadi et al., 2017). para-Nitrosalicylic acid was
converted to its N-hydroxysuccinimidyl ester to activate the carboxylic acid to amidation with diethylglutamate. Subsequent nitro
reduction by hydrogenation afforded the desired amine 1 (Scheme S1). Reductive amination with N-acetylformylpterin yielded the
protected hydroxylated folate 2. 6-Formylpterin was converted to N-acetylformylpterin by refluxing in acetic anhydride. Global de-
protection with sodium hydroxide afforded the desired hydroxylated folate adduct 3.
Compound characterization: 1,5-diethyl 2-[(4-amino-2-hydroxyphenyl) formamido]pentanedioate 1. para-Nitrosalicylic acid
(2.10 g, 11.47 mmol), N-hydroxysuccinimide (1.45 g, 12.62 mmol), and DCC (2.60 g, 12.62 mmol) were added to a 100 mL round-
bottomed flask fitted with a stir bar. DMF (50 mL) was added and stirred overnight. Overnight, a white precipitate had formed.
The reaction was filtered through cotton into a separate 200 mL round-bottomed flask that contained a stirred solution of diethylglu-
tamate HCl (3.03 g, 12.62 mmol), triethylamine (1.28 g, 12.62 mmol), and DMF (20 mL). The solution turned orange upon addition.
After one hour, the DMF was removed in vacuo. The residue was brought up in ethanol (75 mL) and Pd/C (200 mg, 10% Pd) was
added along with a stir bar. The flask was sealed, evacuated, and back-filled with a H2 balloon three times. The reaction stirred over-
night with a H2 balloon to maintain positive pressure. The reaction was diluted with DCM, filtered through a celite plug, and the solvent
was removed in vacuo. A column was run on the residue to afford the title compound as a yellow oil (1.61 g, 41 % three-step yield). 1H
NMR (500 MHz, CDCl3) d 12.40 (s, 1H), 7.27 (d, J = 8.4 Hz, 1H), 7.10 (d, J = 7.1 Hz, 1H), 6.21 – 6.10 (m, 2H), 4.73 (q, J = 7.4 Hz, 1H), 4.26
(q, J = 7.1 Hz, 2H), 4.21 – 4.05 (m, 4H), 2.53 (dt, J = 15.6, 7.1 Hz, 1H), 2.49 – 2.41 (m, 1H), 2.30 (dq, J = 13.2, 6.6, 6.1 Hz, 1H), 2.15 (dt, J =
14.4, 7.2 Hz, 1H), 1.32 (t, J = 7.1 Hz, 3H), 1.25 (t, J = 7.1 Hz, 3H). 13CNMR (126 MHz, CDCl3) d 173.50, 172.04, 170.03, 163.65, 152.51,
127.60, 127.57, 106.47, 104.41, 101.69, 61.82, 60.96, 51.95, 30.54, 27.02, 14.16, 14.13. HRMS (DART, [M+H]+) m/z 339.1555, calcu-
lated for [C16H23N2O6+], 339.1551 (Data S1).
2-[(4-{[(2-amino-4-oxo-1,4-dihydropteridin-6-yl)methyl]amino}-2-hydroxyphenyl)formamido] pentanedioic acid 3. Aryl amine 1
(0.35 g, 1.03 mmol) was dissolved in AcOH (4 mL) and stirred. N-Acetylformyl pterin (0.22 g, 0.94 mmol) was suspended in AcOH
(4 mL) and added slowly to the stirred solution of 1. The solution turned from cloudy to homogeneous within a few minutes. After
15 minutes, a solution of dimethylaminoborane (0.09 g, 1.50 mmol) in AcOH (1 mL) was added and stirred for 10 minutes. The reaction
was heated to 65ꢁC for 20 minutes. The reaction volume was reduced in vacuo to 1 mL and slowly added to a stirred solution of Et2O
(30 mL). A yellow solid crashed out immediately and was collected via filtration. The dried yellow solid was dissolved in NaOH (1.5 mL,
0.5 M) and heated to 70ꢁC for 1.5 h. Once cooled, the solution was acidified to pH 2 by addition of 1 M HCl and stored at 4ꢁC over-
night. The material was isolated via centrifugation. Once the solution was decanted, the solid was washed three times each with wa-
ter, EtOH, and Et2O. The material was dried in vacuo overnight to afford the title compound 3 (0.05 g, 25% two-step yield). 1H
NMR (500 MHz, DMSO-d6) d 12.60 (s, 1H), 12.40 (s, 2H), 11.44 (s, 1H), 8.66 (s, 1H), 8.43 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 8.9 Hz,
1H), 7.06 (t, J = 5.9 Hz, 1H), 6.91 (s, 1H), 6.23 (dd, J = 8.8, 2.0 Hz, 1H), 6.00 (d, J = 2.0 Hz, 1H), 4.47 (d, J = 5.8 Hz, 2H), 4.38 (ddd,
J = 9.6, 7.7, 5.0 Hz, 1H), 2.33 (t, J = 7.4 Hz, 2H), 2.09 (dq, J = 13.0, 7.7 Hz, 1H), 1.94 (ddd, J = 16.7, 14.1, 7.1 Hz, 1H). 13C NMR
(126 MHz, DMSO-d6) d 174.26, 173.76, 170.24, 162.95, 161.32, 157.09, 154.24, 153.54, 149.14, 148.81, 129.41, 128.46, 105.11,
103.64, 98.31, 51.83, 46.24, 30.78, 26.40. HRMS (DART, [M+H]+) m/z 458.1432, calculated for [C19H20N7O7+], 458.1419 (Data S2).
In Vitro Susceptibility Testing
On the day of in vitro testing the drugs were thawed and diluted in modified 7H10 broth to 4-times the maximum concentration to be
tested. The test range for INH was 8 mg/ml to 0.008 mg/ml and the test range for PAS was 64 mg/ml to 0.06 mg/ml. On the day of in vitro
testing the organism was diluted in 7H10 broth to a final concentration of approximately 1 X 105 CFU/ml (in vitro inoculum). Polysty-
rene 96-well round-bottom plates (Corning Inc., Corning, NY) were prepared with 50 ml of 7H10 broth per well. The compounds were
added to the first well prior to being serially 2-fold diluted throughout the row, leaving the last well with 7H10 broth only (growth con-
trol). Fifty ml of the in vitro inoculum was added to each well. Plates were sealed and incubated at 37ꢁC in ambient air for 14-21 days
prior to reading. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of drug required to inhibit growth
of M. tuberculosis observed visually. The MIC assays were run in duplicate. The actual inoculum used was measured by titration in
saline with Tween 80 and plating on 7H10 agar plates (Becton Dickinson, Sparks, MD). The plates were incubated in ambient air at
37ꢁC for 4 weeks.
Transformation, Expression and Purification of Mtb DHFR, Hu DHFR, Mtb Rv2671, and Mtb FDTS
Recombinant plasmids harboring genes encoding Mtb DHFR (in pET-41a(+)),Hu DHFR (in pET-41a(+)) MtbRv2671 (in pET-28a(+)),
and MtbFDTS (in pET-24d) were constructed by GenScript, separately. BL21(DE3) competent E. Coli cells (New England BioLabs)
were transformed with the recombinant plasmids, separately. Transformed cells were grown in LB medium supplemented with
30 mg/mL kanamycin at 37ꢁC until OD600 reached 0.6-0.7. The cells were induced with 1 mM IPTG for 20 hours at 20ꢁC and spun
down at 8000 rpm for 15 minutes. Each gram of wet cell pellet was resuspended in 5 ml of lysis buffer (25 mM Tris pH 8.0,
0.4 M KCl, 5 mM imidazole, 5 mM BME, 5% glycerol, 200 mg/ml lysozyme, 1 mM DNase I). The cell suspension was incubated
for 30-60 minutes at 4ꢁC with gentle rotation followed by sonication until a homogenous lysate was obtained. The lysate was centri-
fuged at 18,000 rpm for 30 minutes and supernatant was collected and filtered through 0.22 mm filter. The Mtb DHFR and Hu
DHFR constructs did not contain histidine tag and were purified over methotrexate-agarose column pre-equilibrated with 4 CV of
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equilibration buffer (20mM Tris-HCl pH 7.5, 50 mM KCl, 2 mM DTT, 0.1 mM EDTA and 15% glycerol). The column was washed with 3
CV of wash buffer (20 mM Tris-HCl pH 7.5, 500 mM KCl, 2 mM DTT, 0.1 mM EDTA and 15% glycerol). The protein was eluted with 3
CV of elution buffer (equilibration buffer pH 8.5 + 2mM DHF). Mtb Rv2671 and Mtb FDTS constructs contained a histidine tag and
were purified using Ni-NTA purification system (Thermo Scientific). The cell lysate supernatant was loaded over the column packed
with HisPur Ni-NTA Superflow Agarose beads and washed with 5 CV of buffer A (25 mM Tris pH 8.0, 0.4 M KCl, 5 mM imidazole, 5 mM
BME and 5% glycerol). The protein was eluted with a gradient of buffer B (25 mM Tris pH 8.0, 0.3 M KCl, 250 mM imidazole, 5 mM
BME, 0.1 mM EDTA and 20% glycerol). For all the purifications, fractions containing DHFR protein were collected, concentrated and
loaded onto a Hi-Prep 26/60 Sephacryl s-200 HR prepacked gel filtration/size exclusion column pre-equilibrated with 1 CV of final
buffer (25 mM Tris pH 8.0, 50 mM KCl, 0.1 mM EDTA, 2 mM DTT and 15% glycerol). The column was washed with another 1 CV of final
buffer and protein elution was monitored with AKTA UV/vis diode array spectrophotometer at 280 nm. Fractions containing pure
enzyme were pooled, concentrated at 10 mg/ml and flash frozen in liquid nitrogen and stored at -80ꢁC.
Enzyme Inhibition Assay for Mtb DHFR, Hu DHFR, and Mtb Rv2671
The DHFR activity of Mtb DHFR, Hu DHFR, and Mtb Rv2671 was measured in 500 ml of assay buffer containing 20 mM TES,
50 mM KCl, 0.5 mM EDTA, 10 mM 2-mercaptoethanol (BME) and 1 mg/ml bovine serum albumin (BSA) with various concentrations
of NADPH and DHF ranging from 0 to 100 mM. The enzyme final concentration of 5-10 nM for MtbDHFR and 10-20 nM for other en-
zymes was used in the assay. Assay was started by adding DHF and monitoring NADPH oxidation at 340 nm. All measurements were
performed at room temperature and in triplicate. Initial velocity data were fit with the Michaelis-Menten equation using Graphpad
Prism 7.0 software. The DHFR activity inhibition assays and IC50 determination were performed in the same assay buffer with
100 mM NADPH and 100 mM DHF. For Mtb Rv2671 50 mM NADPH was used instead of 100 mM. Inhibitors, dissolved in 100%
DMSO, were added to the mixture and incubated for 5 minutes prior to adding DHF.
Jump-Dilution Recovery Assay
Enzyme was incubated with the inhibitor (at concentration 10 times higher than IC50) for 16 hours at 4ꢁC in the absence of substrate to
achieve nearly complete binding of the inhibitor to the DHFR enzyme. The mixture was then diluted 100-fold to stablish the final in-
hibitor concentration well below the IC50 into an assay mixture containing 100 mM DHF and 100 mM NADPH to disturb the equilibrium
condition. Under this condition, minimum residual binding should occur and the observed rate constant, kobs, can be considered as a
reasonable estimate of the dissociation rate constant koff. Enzymatic activity recovery was followed by monitoring NADPH oxidation
at 340 nM for 60 minutes. Progress curves were fit into integrated rate Equation 1 using GraphPad Prism 7.0 using the initial velocity
(the velocity observed in the presence of the inhibitor) as Vi, the steady-state velocity (the final velocity following the dissociation of the
inhibitor) as Vs to yield the observed activity rate constant (kobs). The residence time was estimated as the reciprocal of the observed
activity recovery rate constant, kobs, as described in Equation 2 (Copeland et al., 2011; Walkup et al., 2015). The assay was performed
in triplicate.
À
Á
Vi ꢀ Vs
obst
½Pꢃ = Vst +
1 ꢀ eꢀk
(Equation 1)
kobs
1
1
tR =
=
(Equation 2)
koff kobs
Mtb FDTS NADPH Oxidation Assay
Mtb FDTS NADPH oxidation assay was performed by monitoring NADPH oxidation at 340 nM using Genesys 150 UV-Vis spectro-
photometer from Thermo Scientific. The assay buffer contained 100 mM dUMP, 50 mM FAD, 200 m=mM NADPH, 10 mM mTHF, and
various concentrations of inhibitors (for inhibition assay). The assay was initiated by adding the enzyme at 10 nM final concentration.
The assay was done in triplicate and each reaction was followed for 15 minutes (Hunter et al., 2008). The IC50 was determined by
curve fitting using nonlinear regression and built-in equation ((log) inhibitor vs. response-Standard slope) in GraphPad Prism 7.0.
Crystallization of Mtb DHFR, Data Collection, and Structure Determination
All the crystallization trials were performed by hanging drop vapor diffusion method and using EasyXtal 15-well plates (Qiagen).
MtbDHFR was crystallized using slight modifications of conditions previously reported by Li et al. (4). Pure protein was exchanged
into the same final buffer containing 5% glycerol instead of 15%, incubated with 5 mM NADPH and 2mM ligand for two hours on ice.
An equal volume of this mixture (final protein concentration 5 mg/ml) was mixed with crystallization solution (2 ml + 2 ml) and let to
equilibrate against 500 ml of well solution at 4ꢁC. Large pyramidal crystals grew within 8-12 weeks from solutions comprising
2.1-2.3 M (NH4)2SO4 and 0.1 M sodium acetate pH 4.5.
Crystals were flash frozen in the mother liquor supplemented with 20% glycerol. X-ray data were collected at Stanford Synchrotron
Radiation Light Source (SSRL). Data were indexed and scaled using HKL2000. Structures were solved by molecular replacement
e3 Cell Chemical Biology 26, 1–11.e1–e6, June 20, 2019

Please cite this article in press as: Hajian et al., Drugging the Folate Pathway in Mycobacterium tuberculosis: The Role of Multi-targeting Agents, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.013
using the Phaser (McCoy et al., 2007) and a previously reported complex (PDB ID: 5JA3) (Hajian et al., 2016). The programs Coot
(Emsley and Cowtan, 2004) and Phenix (Adams et al., 2010) were used for structure refinement. Data collection and refinement sta-
tistics are reported in Table S3.
Crystallization of Hu DHFR, Data Collection, and Structure Determination
Hu DHFR was mixed with 10 mM NADPH and 2 mM ligand, incubated on ice for two hours and concentrated to 12 mg/ml. 2 ml of this
solution was mixed with 2 ml of crystallization solutions containing 0.1 M Tris pH 7-7.5, 0.2-0.4 M Li2SO4, 25-35% PEG 4K and 7%
EtOH. Large hexagonal crystals grew within 1-2 weeks at 4ꢁC. Data collection and structure determination was performed similar to
Mtb DHFR. A previously reported Hu DHFR (PDB ID: 4KAK) was used for molecular replacement (Lamb et al., 2013). Data collection
and refinement statistics are reported in Table S3.
Crystallization of Mtb Rv2671, Data Collection, and Structure Determination
Mtb Rv2671 was mixed with 5 mM NADPH and 2 mM ligand, incubated at 4ꢁC overnight and concentrated to 5 mg/ml. 2 ml of crys-
tallization solutions containing 0.1-0.3 M NaCl, 0.1 M Bis-Tris pH 5.5-7.5 and 20-30% PEG 3350 was mixed with 2 ml of protein so-
lution and equilibrated against the well solution at 18ꢁC. Needle crystals grew within 2 weeks. Crystals were flash frozen in the mother
liquor supplemented with 20% glycerol. X-ray data were collected at National Synchrotron Light Source II (NSLS II) at Brookhaven
National Laboratory. Structures were solved by molecular replacement using the Phaser and previously reported complexes (Cheng
and Sacchettini, 2016). The programs Coot and Phenix were used for structure refinement. Data collection and refinement statistics
are reported in Table S3.
Crystallization of Mtb FDTS, Data Collection, and Structure Determination
The ternary complex of FDTS with FAD, dUMP was prepared by mixing the enzyme with a 2-fold molar excess of FAD and a 10-fold
molar excess of dUMP. Prior to crystallization the complex was incubated at 4ꢁC for 18 hours and then concentrated to 12 mg/ml by
ultrafiltration using Amicon Ultracel-10K filters (Millipore). Crystals were obtained by the hanging-drop vapor diffusion technique. An
equal volume of the protein solution was mixed with a reservoir solution containing 18% PEG 8K, 0.15 M MgCl2, 0.1M Na Acetate pH
5.0 and 2 mM DTT, at 4ꢁC. Initially, a shower of small crystals appeared in drops after 1 to 2 days. To delay the onset of crystallization
and to reduce the number of crystals, an oil barrier (100 ml of 1:1 Paraffin: Silicon Oil mix) was applied over the reservoir solution in the
standard hanging drops plates. Because of the favorable kinetics this process provided, larger and well-defined crystals were ob-
tained after a week. Prior to data collection, crystals were soaked in the artificial mother liquor solution supplemented with 20% glyc-
erol (v/v) and cryo-cooled in liquid nitrogen. X-ray data were collected at Stanford Synchrotron Radiation Light Source (SSRL). All
data were integrated and reduced using iMOSFLM (Battye et al., 2011) and scaled using SCALA from the CCP4 suite (Winn et al.,
2011). Data collection and refinement statistics are reported in Table S3.
Selection of PAS-Resistant Mutants
The resistant mutants were generated by growing M. tuberculosis Erdman (ATCC 35801) in modified 7H10 broth (pH 6.6; 7H10 agar
formulation with agar and malachite green omitted) with 10% OADC (oleic acid, albumin, dextrose, catalase) enrichment (BBL Micro-
biology Systems, Cockeysville, MD) and 0.05% Tween 80 with 64 mg/ml of PAS added on a rotary shaker at 37ꢁC for 14 days. After
14 days of incubation 1 ml of the broth was placed onto ten 7H10 agar plates with 10% OADC and 64 mg/ml of PAS. After 4 weeks of
incubation at 37ꢁC several colonies were selected, grown in 7H10 broth with 10% OADC, and tested for PAS resistance in a microtiter
broth dilution assay.
Determination of Mutation Rates
The mutation rates were determined using fluctuation analysis and previously reported methods (Ford et al., 2011, 2013; Huitric et al.,
2010; Pope et al., 2008). Briefly, Mtb cultures were grown to optical density (OD) of 0.7 to 1.0. Approximately 300,000 cells were used
to inoculate 120mL of 7H10 media supplemented with 10% OADC and 0.05% tween 80, giving a total cell count of 10,000 cells per
4mL culture. This volume was immediately divided to 24 starter cultures of 4mL each in 15mL centrifugation tubes. 20 cultures were
grown at 37ꢁC with shaking for 14 days, until reaching an OD of 1.0. Cultures were spun at 4000 RPM for 10 minutes at 4ꢁC and were
resuspended in 250-500mL of the same growth media and spotted onto plates 7H10 agar plates supplemented with 10% OADC and
0.05% tween 80 and the inhibitor concentration at 10X times MIC. Plates were incubated at 37ꢁC for 28 days. Cell counts were deter-
mined by serial dilution of the remaining four cultures. The experiment was performed in triplicate. The mutation rate (m) was calcu-
lated using Equations explained in Quantification and Statistical analysis section.
DNA Extraction from PAS-Resistant Isolates
DNA from the PAS-resistant strains were isolated using TRI Reagent purchased from Sigma Aldrich (St. Louis, MO) and following the
TRI Reagent Protocol. Briefly, the PAS-resistant M. tuberculosis strains were grown in modified 7H10 broth with 10% OADC and
0.05% Tween 80 for 7-10 days. The isolates were diluted to 1 X 107 CFU/ml and 1ml of the isolates was placed into a sterile microfuge
tube. The isolates were centrifuged for 10 minutes at 12,000 rpm. The supernatant was removed, and 1 ml of TRI Reagent was added
to the pellet and vortexed. After standing at room temperature for 10 minutes 0.2 ml of chloroform was added to the tubes. The tubes
were shaken for 15 seconds and allowed to stand for 10-15 minutes. The tubes were centrifuged for 15 minutes at 12,000 rpm at 4ꢁC.
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Please cite this article in press as: Hajian et al., Drugging the Folate Pathway in Mycobacterium tuberculosis: The Role of Multi-targeting Agents, Cell
Chemical Biology (2019), https://doi.org/10.1016/j.chembiol.2019.02.013
The aqueous phase (containing RNA) was removed and discarded. To the interphase and organic phase (DNA and protein) 0.3 ml of
100% ethanol was added, mixed by inversion and allowed to stand for 2-5 minutes. The tubes were centrifuged in a cold room for
5 minutes at 6,000 rpm. The supernatant was discarded and the pellet containing the DNA was washed twice with a 0.1 M trisodium
citrate:10% ethanol solution. Between washing steps, the pellets were allowed to sit at room temperature for 30 minutes and then
centrifuged at 6,000 rpm for 5 minutes. After the final wash the pellet was resuspended in 1 ml of 75% ethanol. Samples were plated
on 7H10 agar to ensure that no viable Mtb remained.
Whole Genome Sequencing
One nanogram of genomic DNA (gDNA) was used for preparing the DNA library using Nextera XT DNA Library Prep Kit (Illumina),
following the manufacturer’s recommended protocol. First, in a skirted PCR plate, gDNA was mixed with 10 ml of tagment DNA buffer
and 5 ml of amplicon tagment mix. The plate was centrifuged at 280xg at 20ꢁC for 1 minute followed by tagmentation program using
thermal cycler setting of 55ꢁC for 5 minutes and hold at 10ꢁC. After the sample reached 10ꢁC, 5 ml of neutralize tagment buffer was
immediately added to neutralize the transposome. The sample was centrifuged at 280xg at 20ꢁC for 1 minute followed by incubation
at room temperature. The tagmented DNA was amplified using a limited-cycle PCR program. Nextera Index 1 (i7) and Index 2 (i5)
adapters and Nextera PCR master mix were added to the tagmented gDNA and the samples were amplified using the following
program on the thermal cycler:
72ꢁC for 3 minutes
95ꢁC for 30 seconds
12 cycles of 95ꢁC for 10 seconds, 55ꢁC for 30 seconds, 72ꢁC for 30 seconds
72ꢁC for 5 minutes
Hold at 10ꢁC.
Library cleanup was performed using AMPure XP beads to remove short library fragments. The PCR products were centrifuged at
280xg at 20ꢁC for 1 minute and the beads were added to each well. The ratio of PCR product to volume of beads was 3:2. The mixture
was shaken at 1800 rpm for 2 minutes followed by incubation at room temperature for 5 minutes. The samples were places on mag-
netic stand and allowed for the liquid to become clear (2-3 minutes). Supernatant was removed and discarded from each well and the
samples were washed with 200 ml of fresh 80% EtOH twice. The supernatant was again removed and discarded by gentle pipetting.
The samples were air-dried on the magnetic stand for 15 minutes and then removed from the stand. The DNA was released from the
beads by adding resuspension buffer followed by shaking at 1800 rpm for 2 minutes. The sample was placed on magnetic stand and
allowed for the liquid to become clear. The supernatant containing clean DNA was transferred to a new plate. The size distribution of
the fragments within the library was checked on an Agilent Technology 2100 Bioanalyzer using a high sensitivity DNA chip. The
libraries were then normalized and pooled for sequencing step. The libraries were sequenced on the MiSeq using v2 2x250 base
pair kit (Illumina). The genome assembly was performed using CLC Genomic Workbench and annotated on the RAST server.
Modelling of PAS-M into Mtb FDTS
Docking procedure was performed using version 2018-1 of the Schrodinger Biologics and Small-Molecule suites (for more details,
¨
¨
see Supplemental Information). All calculations were performed using version 2018-1 of the Schrodinger Biologics and Small-Mole-
cule suites. Solved FTDS in complex with FAD and dUMP was imported into Maestro and prepared with the Protein Preparation
Wizard, which assigns tautomer and ionization states, water orientations, flips asparagine, glutamine, and histidine residues. All
˚
co-crystallization artifacts were removed from the structure, and waters only within 5A of FAD and dUMP were kept (12). All other
parameters were left unchanged on their default setting. Induced fit docking was performed with using the Induced Fit Docking pro-
tocol in Maestro (13). The centroid of the docking grid was generated by identifying the binding pocket residues: (Chain A) Tyr44,
Tyr60, Val67, His69, Ser71; (Chain C) Met198 and His203. Extended sampling was selected, and all other parameters were left un-
changed on their default setting. The ligand used for IFD was PAS-M. The structure was imported into Maestro and prepared for
docking using the LigPrep protocol at pH 7.0 2.0 (14).
Analysis of Lipids and Mycolic Acids
The Mtb Erdman strain was grown by shaking at 37ꢁC in Middlebrook 7H9 broth (Difco) supplemented with albumin-dextrose-cata-
lase and 0.05% Tween 80. At O.D.600 nm of ꢂ 0.32 the culture was divided into 100 ml aliquots and tested compounds dissolved in
DMSO were added in 0.36, 0.72 and 1.44 mg/ml final concentrations for UCP1172 and 0.36 and 0.72 mg/ml final concentrations for
PAS. The final concentration of DMSO in each culture was kept 2 %. The cells grew 24 h statically in the presence of the tested com-
pounds. Then 14C acetate (specific activity 106 mCi/mmol, ARC) was added in the final concentration 1 mCi/ml and the cultures
continued growing for next 24 h. The lipids were extracted from 100 ml culture aliquots as described earlier with minor modifications
(Stadthagen et al., 2005). Briefly, the cultures were transferred to 3 ml chloroform: methanol (2:1) and the mixtures were incubated at
65ꢁC for 2 h. After cooling down, 400 ml of water was added, the mixtures were mixed and centrifuged 15 min at 1 000 x g at room
temperature. The organic phases were removed to the clean tube, dried under N2 and dissolved in 30 ml chloroform: methanol (2:1).
5 ml of each lipid sample were quantified by scintillation counting and 5 ml were loaded on the thin-layer chromatography (TLC)
silica gel plates F254 (Merck). Lipids were separated in chloroform: methanol: water [20:4:0.5] and the plates were exposed to
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Please cite this article in press as: Hajian et al., Drugging the Folate Pathway in Mycobacterium tuberculosis: The Role of Multi-targeting Agents, Cell
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autoradiography film Biomax MR (Kodak) at -80ꢁC for 7 days. Methyl esters of fatty acids (FAME) and mycolic acids (MAME) were
prepared from another 100 ml culture aliquots as previously described (Phetsuksiri et al., 1999). Dried extracts were dissolved in 30 ml
chloroform: methanol (2:1), 5 ml of each sample were quantified by scintillation counting and 5 ml were loaded on TLC plates and
different forms of methyl esters were separated in n-hexane/ethyl acetate [95:5], 3 runs and detected by autoradiography. Alterna-
tively, methyl ester derivatives were loaded on TLC plates impregnated in 5% AgNO3 and activated at 100ꢁC for 1h and separated in
the same solvent. The plates were exposed to autoradiography film Biomax MR (Kodak) at ꢀ80ꢁC for 7 days. The assay was per-
formed in triplicate.
Differential Scanning Calorimetry (DSC)
To determine whether ligand binding to Mtb DHFR is stabilizing or destabilizing, we performed Differential Scanning Calorimetry
(DSC) experiments on 1:1 molar ratio of protein:ligand incubated samples using a Nano-DSC instrument (TA instruments). 600 ml
of Mtb DHFR at a concentration of 12 mM including NADPH and ligands or just NADPH (control) were heated 1ꢁC/min starting at
0ꢁC and up to 120ꢁC. For Mtb FDTS, the protein mixed with FAD and dUMP was used as the control. All samples were degassed
extensively prior to injection into the calorimeter cell in order to prevent formation of air bubbles. The reference cell was filled with
buffer for all runs. A pressure of 3 atm was applied to both cells during the run. The assay was performed in triplicate and the excess
heat capacity scans for the protein transitions were obtained by subtracting a control scan of buffer versus buffer. The data were
corrected for the difference in heat capacity between the initial and the final state by using a sigmoid baseline in the NanoAnalyze
software (TA instruments) and fitted to a two-state transition model to determine the Tm values.
HPLC Analysis of Mtb FDTS Reaction
The reaction mixture was analyzed at different time points on a Shimadzu HPLC system using a reversed phase Phenomenex C 18
column (4.6 x 250 mm, 5 mm), with mobile phase A (5 mM potassium phosphate (pH 7.0), 5 mM tetrabutylammonium dihydrogen
phosphate, and 5% acetonitrile) and mobile phase B (acetonitrile). The flow rate was 1 ml/min and the gradient was as follows:
100% A, 0-25 min; 100-10% A, 25-29 min; 10-100% A, 29-31 min; 100% A, 31-40 min. The injection volume for each sample
was 50 ml and the autosampler was set at 4ꢁC. The standard solution of dUMP and dTMP were run to assign the retention times
and the elution was monitored at 260nm. The experiment was performed in duplicate.
QUANTIFICATION AND STATISTICAL ANALYSIS
The signal intensity for spectrophotometric assay was quantified using Genesys 150 UV-Vis spectrophotometer from Thermo Sci-
entific and the data were analyzed using GraphPad Prism 7.0. All the measurements were performed in triplicate and data shown
in bar graphs are mean STDEV. Thermal stability data was analyzed using the NanoAnalyze software that is associated with the
Nano-DSC instrument (TA instrument) and the data were corrected for the difference in heat capacity between the initial and the final
state by using a sigmoid baseline and fitted to a two-state transition model to determine the Tm values.
The mutation rate (m) was calculated using Equations 3 and 4.
m
m =
(Equation 3)
(Equation 4)
Nt
m = ꢀ lnðP0=PtotÞ
m= number of mutations per culture.
Nt= final number of cells (total CFU).
P0= number of independent cultures with zero mutant colonies.
Ptot= total number of independent cultures.
DATA AND SOFTWARE AVAILABILITY
All the macromolecular crystallography data including the protein sequences, coordinates, and the electron density map are
publically available at the Protein Data Bank (https://www.rcsb.org/) using PDB IDs 6DDP, 6DDS, 6DDW, 6DE4, and 6DE5. PDB
IDs are also provided in Key Resources Table. The genome sequencing data are publically available at NCBI Sequence
Read Archive (https://www.ncbi.nlm.nih.gov/sra/) with the BioProject ID PRJNA523029. The accession numbers are provided in
Key Resources Table.
Cell Chemical Biology 26, 1–11.e1–e6, June 20, 2019 e6