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Synthesis and Structure−Activity Relationships of a New Class of

Oxadiazoles Targeting DprE1 as Antitubercular Agents
Veena D. Yadav,§ Helena I. Boshoff,§ Lena Trifonov, Jose Santinni O. Roma, Thomas R. Ioerger,
Clifton E. Barry, III, and Sangmi Oh*
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ABSTRACT: The continuing prevalence of drug-resistant tuber-

culosis threatens global TB control programs, highlighting the need
to discover new drug candidates to feed the drug development
Downloaded via 14.191.72.181 on September 29, 2023 at 01:34:31 (UTC).

pipeline. In this study, we describe a high-throughput screening hit

(4-benzylpiperidin-1-yl)(1-(5-phenyl-1,3,4-oxadiazol-2-yl)-
piperidin-4-yl)methanone (P1) as a potent antitubercular agent.
Structure−activity guided synthesis led to the discovery of several
analogs with high in vitro potency. P1 was found to have promising
potency against many drug-resistant strains, as well as drug-
susceptible clinical isolates. It also showed cidality against Mtb
growing in host macrophages. Whole genome sequencing of
genomic DNA from resistant mutants raised to P1 revealed
mutations in decaprenylphosphoryl-β-D-ribose 2′-oxidase (DprE1).
This novel oxadiazole scaffold expands the set of chemical tools for
targeting a well-validated pathway to treat tuberculosis.
KEYWORDS: 1,3,4-oxadiazole, structure−activity relationship, mycobacteria, tuberculosis, multidrug resistance, high-throughput screening

T he goal of the World Health Organization to eliminate

tuberculosis (TB) by the year 2050 was undermined by the
COVID-19 pandemic with increases in numbers of cases,
numbers of deaths, and incidence of drug resistant cases
recorded from 2020 to 2022.1,2 Continuing rise and spread of
drug resistant tuberculosis is a global health threat and increases
pressure on drug discovery programs to deliver new candidates
to expand the TB drug development pipeline.
Since the mycobacterial cell wall is an essential protective
barrier against various cellular stresses and important for
virulence in the host, it has been the focus of countless efforts
to identify small-molecule inhibitors of enzymes involved in its
biogenesis.3 The vulnerability of drug targets in the cell wall has
been validated by the chemotherapeutic efficacy of several
antitubercular drugs in patients, including isoniazid and
ethambutol, which are part of the first-line antitubercular drug
regimen. Many antitubercular agents inhibiting mycolyl-
arabinogalactan-peptidoglycan (mAGP) biogenesis have been
introduced of which several have demonstrated therapeutic
potential based on their in vivo efficacy.4,5 The well-studied
protein targets include not only the NADH-dependent enoyl-
acyl carrier protein reductase (InhA) which is the target of
isoniazid but also the mycobacterial membrane protein Large 3
(MmpL3) and the decaprenylphosphoryl-β-D-ribose 2′-oxidase
(DprE1). As an inhibitor of MmpL3, ethambutol-inspired
SQ109 is in clinical trials with its bactericidal activity against
© 2023 The Authors. Published by
American Chemical Society
1275
drug-susceptible and drug-resistant TB strains ascribed to its
inhibition of translocation of trehalose monomycolate (TMM)
across the membrane.6 In the case of DprE1 inhibitors, four drug
candidates with three scaffolds, BTZ-043, macozinone (PBTZ-
169), quabodepistat (OPC-167832), and TBA-7371, are in the
different phases of clinical trials.7 BTZ-043 and macozinone are
prodrugs with a benzothiazinone core, which are activated by
DprE1 resulting in covalent suicide inhibition of the enzyme.8 In
contrast, quabodepistat and TBA-7371 are noncovalent
inhibitors of DprE1 with different structural features, a
carbostyril and a 1,4-azaindole scaffold, respectively, and good
therapeutic profiles.9 Since several structurally diverse inhibitors
of MmpL3 and DprE1 have been identified, of which several are
in different stages of the TB drug development pipeline, the
concern has been raised that these are promiscuous drug
targets.10 Nevertheless, the inhibition of bacterial growth along
with the in vivo efficacy of several of these inhibitors confirms the
vulnerability of these targets and supports efforts to assess and
develop such inhibitors.
Received: July 5, 2023
Accepted: August 9, 2023
Published: August 15, 2023
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In an effort to discover new antimicrobial agents, screening of Scheme 1. General Synthetic Scheme of P1 and Its
the 80,000 compound Janssen Jump-stARter Molecular Derivatives P2−P5, P8−P10, and P12−P30a
Library11 against Mtb growing on glucose or on dipalmitoyl-
phosphatidylcholine/cholesterol as carbon sources yielded a
1,3,4-oxadiazole-containing hit, P1 (Figure 1), which inhibited
Mtb growth under both growth conditions. The oxadiazole core
represents a promising new chemotype for antitubercular drug
discovery and has served as a privileged scaffold in several drug
discovery programs serving as linker or a key pharmacophoric
moiety in several approved as well as investigational stage
drugs.12 For example, a new class of non-β-lactam antibiotics
targeting cell-wall biosynthesis contained an oxadiazole
moeity.13 In addition, an oxadiazole can serve as a bioisosteric
a
substitution for the hydrazide moiety, such as that found in the Reagents and conditions: (a) K2CO3 (2.0 equiv), DMF, rt, 18 h; (b)
first-line anti-TB drug isoniazid.14 Some oxadiazole derivatives NaOH (2.5 equiv), MeOH, 50 °C, 1.5 h; (c) EH, EDC, HOBt,
DIPEA, 0 °C to rt, DCM, 9−14 h, 75−81%; Letters A, C, and E in
red indicate each moiety depicted in Figure 1.

Scheme 2. Synthesis of P6 and P7a

Figure 1. Chemical structure of hit compound P1 and its derivatization

for the SAR study.

Table 1. Biological Screening of P1

MIC (μM) Reporter gene assay a
Reagents and conditions: (a) methylpiperidine-4-carboxylate (1.2
cell DNA equiv), K2CO3 (2.0 equiv), DMF, 110 °C, 19 h, 81%; (b)
Hit 7H9a GBSAb DPPCc Chold CydKOe wallf damageg
methylpiperidine-4-carboxylate (1.2 equiv), K2CO3 (2.0 equiv),
P1 0.78 0.78 0.6 0.78 0.6 yes no DMF, 55 °C, 25 h, 42%; (c) NaOH (2.5 equiv), MeOH, 50 °C,
INH 0.2 0.3 0.2 0.39 0.39 NDh no 1.5 h; (d) 4-benzylpiperidine, EDC, HOBt, DIPEA, 0 °C to rt, DCM,
a 12 h, P6 (79%) and P7 (81%).
MIC of compound against Mtb strain H37Rv in Middlebrook 7H9/
ADC/Tween 80. bMIC of compound against Mtb strain H37Rv in
Middlebrook 7H9/BSA/Tyloxapol with glucose (GBSA) or dipalmi- Scheme 3. Synthesis of P11a
toylphosphatidylcholine (DBSA) as the carbon source. cMIC of
compound against Mtb strain H37Rv in Middlebrook 7H9/DPPC/
cholesterol/BSA/Tyloxapol. dMIC of compound against Mtb strain
H37Rv in Middlebrook 7H9/cholesterol/BSA/Tyloxapol. eMIC of
compound against Mtb strain H37Rv in Middlebrook 7H9/ADC/
Tween 80 with cydC::aph. fInduction of the cell wall responsive
iniBAC promoter as measured using the piniB-LUX reporter strain.
g
Induction of the DNA damage responsive recA and radA reporters
using the precA-LUX and pradA-LUX reporter strains. hNot
determined.

have also been reported to interact with novel Mtb targets.12,14,15

Previously, GSK reported the SAR of a 1,3,4-thiadiazole-
containing hit (GSK710) that was selected from the TB-set with
enzymatic activity against DprE1, which shared high degree of
structural similarity with P1.16,17 Based on these favorable
properties, P1 was selected for formal hit assessment.
Determination of the concentration required to fully inhibit
growth of Mtb (minimum inhibitory concentration, MIC)
confirmed that the compound was active, with sub-micromolar
MIC, against Mtb growing on both glycolytic (glucose) and
gluconeogenic (dipalmitoylphosphatidylcholine and cholester-
ol) carbon sources (Table 1). Profiling of the compound in
several reporter assays revealed that the compound perturbed
1276
a
Reagents and conditions: (a) 4-benzylpiperidine (1.2 equiv), NEt3
(2.0 equiv), DCM, 2 h, 72%; (b) TFA/DCM (1/4), 2 h; (c) K2CO3
(2.0 equiv), DMF, rt, 18 h, 52%.
cell-wall synthesis, but was unlikely to result in DNA damage or
respiratory inhibition (Table 1), based on phenotypic assays for
(1) inhibition of cell wall mycolyl-arabinogalactan biosynthesis,
(2) respiration through the bc1 menaquinol-cytochrome c
oxidoreductase, or (3) DNA damage (Table 1).18,19
We investigated the structure−activity relationships (SAR) of
the initial hit P1 in five regions as shown in Figure 1. General
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Table 2. Structural Aspects and Antitubercular Activities of P1−P10

a
MIC of compounds tested against Mtb H37Rv in Middlebrook 7H9/glucose/casitone/tyloxapol. bMIC of compounds tested against Mtb H37Rv
in 7H9/glucose/BSA/tyloxapol. cCytotoxicity of compound tested against HepG2 cells in DMEM/10% FBS supplemented with glucose.
d
Cytotoxicity of compound tested against HepG2 cells in DMEM/10% FBS supplemented with galactose. eNot determined.

synthesis of 1,3,4-oxadiazole analogs (P2−P5, P8−P10, P12−
P30) including P1 started from a coupling reaction of
commercially available 2-(methylsulfonyl)-1,3,4-oxadiazoles
(1) with appropriate cyclic amines (2) to generate intermediate
3. After saponification of the ester group, the resulting carboxylic
acid was coupled with appropriate amine to form the desired
product (Scheme 1).20−23
The 1,3,4-thiadiazole and 1,2,4-oxadiazole-based analogs (P6
and P7) were prepared from commercially available 2-chloro-5-
phenyl-1,3,4-thiadiazole (4a) and 3-bromo-5-phenyl-1,2,4-
oxadiazole (4b) via nucleophilic substitution with piperidine-
4-carboxylate to afford the corresponding intermediates 5a and
5b.24 Further basic hydrolysis afforded the corresponding acids,
which were used directly for further EDC coupling with amine 4-
benzylpiperidine to afford compounds P6 and P7 (Scheme 2).
P11 with a sulfonyl linker instead of the carbonyl group was
prepared from commercially available Boc-protected 4-
(chlorosulfonyl)piperidine (6) and 4-benzylpiperidine. After
the coupling reaction followed by deprotection of the Boc group,
the same amide coupling reaction with 4-benzylpiperidine
afforded the final compound P11 (Scheme 3).
This focused library of 29 analogs was evaluated for
antitubercular activity by MIC determination against Mtb in
two different media, specifically media with or without albumin
in the presence of glucose as carbon source (GBSA and GCas,
respectively) to assess the effect of protein binding on the
activity. The frontline antitubercular agent, isoniazid, served as
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the positive control (Tables 2 and 3). Compounds P2−P5 were
synthesized to evaluate the importance of the A ring on the left-
hand side. Compared to the initial hit, P1 (MIC = 0.1 μM),
which has a phenyl ring at this position, replacement with a
methyl group in P2 caused the complete loss of activity, whereas
a pyridine in P3 caused a 4-fold increase in MIC value (0.39
μM), indicating that the hydrophobic and aromatic phenyl ring
was essential for activity. Substitution of the para-position of the
A ring with a small, electron-withdrawing fluoro substituent
(P4) was tolerated, but a trifluoromethyl substituent (P5) at the
same position eliminated its activity against Mtb, showing the
importance of the substituent size on the A ring.
The importance of the heteroatoms in the oxadiazole ring
(core B) was explored via synthesis of P6 whereas the
regioisomeric contribution of the atoms was evaluated through
synthesis of P7. Exchange of the oxygen atom in the oxadiazole
ring with a sulfur atom to form a thiadiazole ring (P6) and
modification of the 1,3,4-oxadiazole to a 1,2,4-oxadiazole moiety
(P7) were tolerated (MICs = 0.3 μM) with only a slight increase
in MIC compared to P1. These results suggest that three
heteroatoms with a lone pair of electrons are important by
functioning as hydrogen-bond acceptors within the binding
pocket of the target protein, although the limited number of
compounds evaluated in the assessment of core B limits any
strong conclusions. To explore the role of the piperidine ring in
part C, a different regioisomer (P8), a different ring size with a
pyrrolidine analog (P9), and a methyl insertion between the
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Table 3. Structural Aspects and Antitubercular Activities of P11−P30

a
MIC of compounds tested against Mtb H37Rv in Middlebrook 7H9/glucose/casitone/tyloxapol. bMIC of compounds tested against Mtb H37Rv
in 7H9/glucose/BSA/tyloxapol. cCytotoxicity of compound tested against HepG2 cells in DMEM/10% FBS supplemented with glucose.
d
Cytotoxicity of compound tested against HepG2 cells in DMEM/10% FBS supplemented with galactose. eNot determined.

piperidine ring C and the carbonyl carbon (P10) were
synthesized, all of which resulted in loss of potency. For all
active analogs described in Table 2 including the initial hit, P1
(MIC = 0.10 μM in GCas, 0.78 μM in GBSA), protein binding
was detrimental to whole cell activity as evidenced by the 3−8-
fold increase in MIC in protein-enriched (GBSA) media.
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The carbonyl group between parts C and E was critical
because replacement of the carbonyl with a sulfonyl group was
not tolerated (Table 3: P11, MIC = 12.5 μM).
For the SAR study of the right-hand side part E, compounds
P12−P30 were synthesized to evaluate the benzyl-substituted
piperidine moiety. Replacement of the piperidine with a
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Figure 2. P1 is cytotoxic against Mtb growing in macrophages. One day
Mtb-infected J774 macrophages were treated for 7 days with the
indicated compounds followed by colony forming unit enumeration on
solid medium.
piperazine (P12 and P13) or with an aliphatic amine (P14 and
P15) abolished whole cell activity. Although P16 without the
benzyl group had complete loss of activity, simple aliphatic
substituents like trifluoromethyl (P17), tert-butyl (P18), and
isopropyl (P19) still showed moderate MICs in GCas medium
with 2−8-fold higher MICs in GBSA media, suggesting a similar
pattern on effect of protein binding as well as the importance of a
hydrophilic substituent on the piperidine ring. Interestingly, P20
with an embedded tetrahydroisoquinoline moiety as a fused
bicyclic analogue had diminished activity, whereas its rotatable
analogue, P21, with a phenyl conjugated piperidine, was
modestly active against Mtb cells. The 20-fold loss of activity
associated with replacement of the benzyl group of P1 (MIC =
0.10 μM) with a phenyl group in P21 (MIC = 2.3 μM)
confirmed the importance of the conformational flexibility
generated by the methyl linker between the piperidine and
phenyl rings. In addition, the 4-benzylpiperidine moiety was
crucial in terms of regioisomeric aspects as well as ring size
because the regioisomer P22 with a 3-benzylpiperidine moiety
was inactive, and replacement of the piperidine with a smaller
pyrrolidine, P23, reduced its activity about 60-fold (MIC = 6.25
μM). Replacement of the piperidine ring with a cyclohexyl
amine (P24) resulted in an almost 50-fold reduction in activity
(MIC = 4.7 μM) compared to P1 with protein binding of P24 in
GBSA contributing to complete lack of whole cell activity.
Analogs P26−P29 were prepared to further examine the methyl
linker between piperidine and phenyl ring, which demonstrated
that conformation restriction due to unsaturation (P26) or a
methyl substitution in the benzylic position (P27) did not
impact whole cell activity (MIC = 0.30 and 0.20 μM,
respectively) whereas the introduction of oxygen (P28, MIC
= 2.3 μM) or replacement of the sp3 carbon with a carbonyl
group (P29, MIC = 4.7 μM) was detrimental for activity, as
evidenced by a 20−50-fold decrease in potency compared to P1.
Notably, replacement of the aromatic benzene with an
aliphatic cyclohexane in P25 improved the potency and reduced
the detrimental effect of protein binding on whole cell activity
(MIC = 0.05 μM in GCas and 0.20 μM in GBSA). Similarly,
P30, which has a trifluoromethyl substituent in the para position
on the phenyl ring, was equipotent to P1 but its activity was only
3-fold lower in the presence of protein in the media (MIC = 0.10
μM in GCas and 0.30 μM in GBSA). Based on this preliminary
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SAR, further modification of part E with different heteroaryls,
aliphatic cycles, and different substituents on the benzene ring
would allow the optimization of compounds that retain potency
in the presence of protein which is essential for in vivo proof of
concept studies. Analysis of the correlation between MIC in
either medium and cLogP for compounds P1 and P11−P30
based on the assumption that rings A, B, and C formed the key
pharmacophore revealed no correlation between whole cell
activity and hydrophobicity. Moreover, there was no correlation
between cLogP and the effect of protein binding as measured by
the fold difference between MIC values in protein-free and
-supplemented media suggesting that BSA binding and access to
the cellular target were not driven by hydrophobicity in contrast
to reported correlates between cLogP and whole cell activity
(Figure S1).25 This lack of correlation was unaffected by
including all compounds with whole cell activity. The
compounds had an excellent selectivity index as evidenced by
the lack of cytotoxicity against HepG2 cells as well as lack of
overt mitochondrial toxicity as seen by lack of cytotoxicity
during growth of these cells on galactose as a carbon source
(Table 2).26
The efficacy of P1 against Mtb growing in host macrophages
revealed that the compound exerted remarkable cidality at 5-fold
the MIC value, even exceeding that of rifampicin (Figure 2).
This suggested that the compound has access to the phagosomal
environment of Mtb in the host and that the target pathway
remained vulnerable during host pathogenesis. In addition, the
level of pre-existing resistance in a panel of clinical isolates
including several multidrug and extensively drug resistant strains
was low with 5 strains exhibiting low (approximately 4−5-fold)
levels of resistance (Table 4), suggesting that resistance
mechanisms for clinically used drugs pose limited concern in
potential drug development efforts for this scaffold.
To gain an understanding of the mechanism of action of P1,
resistant mutants were generated on solid growth medium
containing 5- and 10-fold the liquid MIC concentration of P1.
Colonies grew up at frequencies of 3 × 10−8 cells at 5-fold MIC
concentrations, whereas no resistant mutants could be
generated at higher compound concentrations, even when
plating 109 cells. All colonies were found to be 4−16-fold
resistant to P1 as measured by liquid MIC determinations.
Whole genome sequencing of 7 mutants revealed that 6
contained a T to C single nucleotide polymorphism (SNP) at
position 4237005 of the Mtb H37Rv genome (re-sequenced)
corresponding to a Leu368Pro mutation in DprE1, the
decaprenylphosphoryl-β-D-ribose 2′-oxidase essential for cell
wall arabinan biosynthesis of mycobacteria (Table 5). Leu368 is
13.5 Å away (Cβ to Cβ) from the Cys387 residue to which
benzothiazinones covalently attach in the active site (such as
BTZ043, see Figure 3). Leu368 is oriented away from the active
site pocket toward the surface on the other side of the protein.
However, Leu368 occurs at the terminus of a β-strand that is
adjacent to the β-strand containing Cys387, and other residues
adjacent to Leu368 (Lys367 and Val365) line the pocket and
make contact with the inhibitor. We speculate that mutating
Leu368 to proline at the end of this β-strand might induce a
backbone shift that propagates to the adjacent residues in the
strand, thereby prohibiting binding of P1 (assuming it binds in
the same pocket) while maintaining catalytic activity. Another
mutant had an A to G SNP at position 4269441 resulting in a
Ser173Pro mutation in UbiA, the decaprenylphosphoryl-5-
phosphoribose synthase, which functions upstream of DprE1
(Table 5). This mutation has been previously identified in
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Table 4. Activity of P1 against Drug-Susceptible and Drug-Resistant Mtb Strains

MIC (μM) MIC (μM)
Strain DSTa BDQ P1 Strain DSTa BDQ P1
Drug-Susceptible Strains
H37Rv ATCC27294 0.15 0.6 K11b00DS 0.3 1.56
Erdman 0.1 0.39 K12b00DS 0.04 1.56
HN878 0.05 0.78 K13b00DS 0.1 1.2
CDC1551 0.07 0.6 K14b00DS 0.6 1.2
CRC clinical strain 0.2 2.3 K15b00DS 0.07 0.78
K5072429
K04b00DS 0.04 0.78 K16b00DS 0.1 0.78
K05b00DS 0.05 1.2 K17b00DS 0.07 0.78
K07b00DS 0.15 1.2 K35b00DS 0.07 1.2
K09b00DS 0.2 1.2 NIH_G12 0.05 0.78
K10b00DS 0.1 1.2
Drug-Resistant Strains
NIH_G11R ECs 0.15 0.6 NIH1B21 HEth 0.07 1.56
NIH_G145 Eth 0.2 1.2 NIH1B314 HEThCsOThLZ <0.02 3.13
NIH_G169 SCs 0.15 0.39 NIHA38 HTh 0.05 0.39
NIH_G17R HEthCs 0.1 0.6 NIHB140 HTh 0.05 0.6
NIH1B127 HOTh 0.1 0.39 NIHB43 RHSTh 0.05 0.78
Multidrug-Resistant (MDR) Strains
CRC clinical strain 01291696 HRS 0.05 0.78 NIH_G21R HRERbCs 0.02 2.3
K18b01MR HRERb 0.04 1.56 NIH_G22R HRERb 0.1 1.2
K21b00MR HRES 0.07 1.2 NIH_G269DR HRERb 0.1 1.2
K22b00MR HRERb 0.15 1.56 NIH_G79 HRSEth 0.07 1.56
K25b00MR HREZRbTh 0.1 1.2 NIHA37 HRRb 0.05 0.39
K26b00MR HREZRb 0.1 1.2 NIHA48 HREthTh 0.1 1.56
K29b00MR HRSPO 0.2 1.2 NIHB302 HREZRbTh 0.04 0.78
NIH_G10R HRERb 0.1 0.6 NIHB67 HRPOTh 0.3 0.78
NIH_G19R HRESCsRb 0.1 1.56 NIHB68 HRPZThRb 0.1 0.39
MDR+ Strains
K20b00MR HREZSK <0.02 4.7 NIH_G367DR HRXM <0.02 3.13
K32b00MR HRKZCpmAmkThCs 0.1 1.56 NIH_G76MR HRXM <0.02 1.2
K33b00MR HREZSKPTh 0.07 2.3 NIHB188 HRZThRbOML 0.04 0.6
Kb019 HREPKOTh 0.07 4.7 NIHB270 HREZThOLRb 0.07 1.56
Extensively Drug-Resistant (XDR) Strains
026 K111 HRESKPCpmAmkThCsOM 0.2 1.56 NIH1B178 HRSEKThCsPOL 0.05 0.78
LRbZLzd
028K111 HRESKPCpmAmkThCsOM 0.15 1.56 NIH1B250 HRESPORbZThCsMLRbCpm 0.07 2.3
LRbZLzd
CRC clinical strain 00202293 HRESKPEthML 0.02 1.56 NIHB13 HRESKPThCsOMLAmkCpmTh 0.05 0.78
K37b00XR HRKSXML 0.1 3.13
a
DST, drug resistance profile as determined by drug sensitivity testing. Amk, amikacin; Cpm, capreomycin; Cs, D-cycloserine; E, ethambutol; Eth,
ethionamide; H, isoniazid; K, kanamycin; L, levofloxacin; Lzd, linezolid; M, moxifloxacin; O, ofloxacin; P, para-aminosalicylic acid; R, rifampicin;
Rb, rifabutin; S, streptomycin; Th, prothionamide; Z, pyrazinamide.

Table 5. Mutations Observed in P1-Resistant Mutants

P1-Resistant Strain (Fold Resistance)
SNP position in chromosome (gene/protein) H37Rv A1 (6×) A4 (8×) B5 (8×) B6 (8×) C2 (8×) C4 (16×) D4 (4×)
4237005 (Rv3790/DprE1:L368P) T C C C C C C -
4269441 (Rv3806c/UbiA:S173P) A - - - - - - G
6684 (Rv0005/GyrB:R482L) G - - - - - - -
976904 (Rv0878c/PPE13:+1) T TG TG TG TG TG TG -
2181405 (Rv1929c/Rv1929c:T172S) T - - - - - A -
3244329 (Rv2930/FadD26:+1) C CA CA CA CA CA CA CA
3380454 (intergenic:+1) G - GC GC GC - GC GC
3537818 (Rv3169/Rv3169:A194P) G - - - - - - C
3558903 (3558902:T>A) T - - - - - A -
4124644 (Rv3683/Rv3683:V35A) T C C C C C C C

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Figure 3. Proximity of L368 to C387 in the active site of DprE1 (PDB:
6HEZ). The benzothiazinone inhibitor BTZ043 is covalently attached
to C387 (green). L368 (yellow) is oriented outside of the pocket, but
the adjacent residues in the same β-strand (K367 and V365) line the
active site pocket and contact the inhibitor. Mutating L368 to proline
could cause a backbone shift affecting these neighboring residues in the
β-strand (blue), which might abrogate binding of the oxadiazole
inhibitor P1, providing a structural hypothesis for the mechanism of
resistance.
ethambutol resistant Mtb strains and likely affects flux through
the arabinan biosynthetic pathway.27 Although several other
SNPs were identified in the P1-resistant mutants, several have
been found to convey low-level nonspecific resistance to a
variety of inhibitors, including an insertion in fadD26 encoding a
fatty-acid-AMP ligase involved in PDIM biosynthesis likely
affecting cell wall permeability and an insertion in ppe13 which
may affect porin-mediated uptake of the compound, which likely
contribute to the range of resistance profiles (6−16-fold
resistance) observed between the strains with dprE1 poly-
morphisms (Table 5 and Table S1). In contrast, ubiA conferred
lower levels of resistance, as previously observed with
ethambutol.27 We confirmed that mutations in UbiA raised to
other cell wall targeting inhibitors (unpublished data) also
conferred low to moderate resistance whereas certain mutations
that have been observed to confer resistance to other DprE1-
targeting compounds including gave a diversity of resistance
profiles with some mutations (Gly248Ser, Tyr314Cys, Pro116S-
er) conferring resistance while strains with other mutations
(Tyr314His, Asn364Ser) remained susceptible.28−31 This
strongly suggests that P1 likely targets the decaprenylphos-
phoryl-β-D-ribose 2′-oxidase, which is further corroborated by
the positive response of our cell wall reporter assay.
In summary, screening of the 80,000 compound Janssen
Jump-stARter Molecular Library yielded a phenyl oxadiazole
piperidine scaffold with promising in vitro and intramacrophage
activity against Mtb. Synthesis of a focused library of 29 analogs
allowed us to identify the salient features on the scaffold that are
essential for future drug development efforts on this hit. The
identification of DprE1 as the likely cell wall biosynthetic target
aligns with the known vulnerability of the arabinan biosynthetic
pathway. There are several DprE1-targeting scaffolds currently
1281
pubs.acs.org/acsmedchemlett Letter
under clinical evaluation for TB chemotherapy, and this phenyl
oxadiazole piperidine adds a novel chemotype for future
evaluation as a cell wall biosynthetic inhibitor in the TB drug
development pipeline.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.3c00295.
Supporting table and figure, experimental details for
chemistry and biology, characterization data for synthetic
compounds including the copies of NMR (1H and 13C)
and mass (HRMS and UV) data (PDF)
■ AUTHOR INFORMATION
Corresponding Author
Sangmi Oh − Tuberculosis Research Section, Laboratory of
Clinical Immunology and Microbiology, National Institute of
Allergy and Infectious Diseases (NIAID), National Institutes of
Health (NIH), Bethesda, Maryland 20892, United States;
orcid.org/0000-0003-0895-1110; Phone: +1-301-761-
6401; Email: sangmi.oh@nih.gov; Fax: +1-301-480-3506
Authors
Veena D. Yadav − Tuberculosis Research Section, Laboratory of
Clinical Immunology and Microbiology, National Institute of
Allergy and Infectious Diseases (NIAID), National Institutes of
Health (NIH), Bethesda, Maryland 20892, United States
Helena I. Boshoff − Tuberculosis Research Section, Laboratory
of Clinical Immunology and Microbiology, National Institute
of Allergy and Infectious Diseases (NIAID), National Institutes
of Health (NIH), Bethesda, Maryland 20892, United States;
orcid.org/0000-0002-4333-206X
Lena Trifonov − Tuberculosis Research Section, Laboratory of
Clinical Immunology and Microbiology, National Institute of
Allergy and Infectious Diseases (NIAID), National Institutes of
Health (NIH), Bethesda, Maryland 20892, United States
Jose Santinni O. Roma − Tuberculosis Research Section,
Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Diseases (NIAID),
National Institutes of Health (NIH), Bethesda, Maryland
20892, United States
Thomas R. Ioerger − Department of Computer Science and
Engineering, Texas A&M University, College Station, Texas
77843, United States
Clifton E. Barry, III − Tuberculosis Research Section,
Laboratory of Clinical Immunology and Microbiology,
National Institute of Allergy and Infectious Diseases (NIAID),
National Institutes of Health (NIH), Bethesda, Maryland
20892, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.3c00295
Author Contributions
§
V.D.Y. and H.I.B. contributed equally.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was supported by a grant from the Bill & Melinda
Gates Foundation (INV-001727) and in part by the Division of
Intramural Research, NIAID, NIH. We thank BIO Ventures for
https://doi.org/10.1021/acsmedchemlett.3c00295
ACS Med. Chem. Lett. 2023, 14, 1275−1283
ACS Medicinal Chemistry Letters
Global Health (BVGH) for facilitating a collaboration with
Johnson & Johnson for the sharing of the Janssen Jump-stARter
Molecular Library.
■ ABBREVIATIONS
Mtb, Mycobacterium tuberculosis; TB, tuberculosis; SAR,
structure−activity relationship; MDR, multidrug-resistant;
XDR, extensively drug-resistant; mAGP, mycolyl-arabinogalac-
tan-peptidoglycan; InhA, NADH-dependent enoyl−acyl carrier
protein reductase; MmpL3, mycobacterial membrane protein
Large 3; DprE1, decaprenylphosphoryl-β-D-ribose 2′-oxidase;
MIC, minimum inhibitory concentration; SNP, single nucleo-
tide polymorphism; INH, isoniazid; BDQ, bedaquiline; DMSO,
dimethylsulfoxide; DMF, dimethylformamide; EDC, 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide hydrochloride; HOBt,
N-hydroxybenzotriazole; DIPEA, N,N-diisopropylethylamine;
DCM, dichloromethane; TFA, trifluoroacetic acid
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