Abstract
A potent, non-cytotoxic indazole sulfonamide was identified by
high-throughput screening of >100,000 synthetic compounds for activity
against Mycobacterium tuberculosis (Mtb). This non-cytotoxic compound
did not directly inhibit cell wall biogenesis but triggered a slow
lysis of Mtb cells as measured by release of intracellular green
fluorescent protein (GFP). Isolation of resistant mutants followed by
whole-genome sequencing showed an unusual gene amplification of a 40
gene region spanning Rv3371 to Rv3411c and in one case a potential
promoter mutation upstream of guaB2 (Rv3411c) encoding inosine
monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation
confirmed direct inhibition of IMPDH by an uncompetitive mode of
inhibition and growth inhibition could be rescued by supplementation
with guanine, a bypass mechanism for the IMPDH pathway. Beads
containing immobilized indazole sulfonamides specifically interacted
with IMPDH in cell lysates. X-ray crystallography of the
IMPDH-IMP-inhibitor complex revealed that the primary interactions of
these compounds with IMPDH were direct pi-pi interactions with the IMP
substrate. Advanced lead compounds in this series with acceptable
pharmacokinetic properties failed to show efficacy in acute or chronic
murine models of tuberculosis (TB). Time-kill experiments in vitro
suggest that sustained exposure to drug concentrations above MIC for 24
hours were required for a cidal effect, levels that have been difficult
to achieve in vivo. Direct measurement of guanine levels in resected
lung tissue from tuberculosis infected animals and patients revealed
0.5–2 mM concentrations in caseum and normal lung tissue. The high
lesional levels of guanine and the slow lytic, growth-rate dependent,
effect of IMPDH inhibition pose challenges to developing drugs against
this target for use in treating TB.
Keywords: Target validation, IMPDH, guanine, purine salvage,
Mycobacterium tuberculosis, indazole sulfonamide
INTRODUCTION
As the incidence of drug-resistant tuberculosis (TB) continues to
worsen, there is a pressing need for new agents to treat this
recalcitrant disease ^[132]1. One of the key drivers of drug-resistance
is the lengthy 6-month course of therapy that must be completed to
achieve sterile cure in patients ^[133]2, [134]3. Therefore many
current TB drug discovery programs focus on strategies to reduce
treatment duration, often by prioritizing efforts to inhibit targets
other than those already inhibited by current front-line therapies.
Because of the historical difficulty of translating the products of
target-based medicinal chemistry into compounds with whole-cell
activity in the antibacterial field, many drug discovery efforts begin
with the target-agnostic process of whole-cell screening for growth
inhibition ^[135]4.
Series with whole-cell potency against Mtb resulting from such
screening programs offer an attractive starting point for lead
optimization efforts but understanding target novelty requires
deconvolution of the molecular mechanism of cell death induced by that
series. We have previously proposed whole genome sequencing of
resistant mutants as a scalable technique to identify SNPs within
potential targets and shown that this works with a small set of
screening hits ^[136]5. This methodology works well for small molecule
hits that directly interact with a single protein target but screening
hits may, of course, have a more complex mechanism. Even where a single
enzyme targeted by a single inhibitor is the predominant mechanism of
inhibition of cell growth, whole genome sequencing sometimes gives
surprising results that do not immediately provide a clear candidate
for the protein target.
Mtb possesses the enzymatic machinery to either synthesize purine
nucleotides de novo or scavenge them from the host to provide the
essential nucleotides required for DNA synthesis ^[137]6, [138]7. Mtb
was the first bacterium from which an adenosine kinase activity was
identified and low local concentrations of adenosine are thought to be
a feature of at least some tuberculous lesion types ^[139]8, [140]9.
The de novo biosynthetic pathway for guanine-containing nucleotides as
well as the salvage pathways of purine nucleotides that yield inosine
and hypoxanthine as intermediates ([141]Fig. 1), all pass through a
common intermediate, inosine 5’-monophosphate, to derive the required
guanine and adenine containing deoxynucleotide precursors for DNA
synthesis. Guanine-containing precursors in particular require
conversion of inosine 5’-monophosphate to xanthine 5’-monophosphate
through the action of inosine 5’-monophosphate dehydrogenase (IMPDH).
Mtb encodes three apparent homologs of IMPDH on its chromosome (guaB1,
guaB2 and guaB3) but only one (guaB2, Rv3411c) has been shown to be
essential and to catalyze the NAD^+-dependent dehydrogenation and
hydrolysis of inosine 5’-monophosphate to xanthine 5’-monophosphate
^[142]10, [143]11. Several series of small molecule inhibitors of IMPDH
have been developed and recently crystal structures of a truncated form
of the Mtb enzyme in complex with these inhibitors have appeared
^[144]10, [145]12–[146]14. In general the whole cell activity of these
inhibitors has been only in the 1–10 µM range.
Figure 1. Purine salvage pathway.
[147]Figure 1
[148]Open in a new tab
IMP, Inosine monophosphate; GMPS, guanosine monophosphate synthase;
XMP, Xanthosine monophosphate; GMP, guanine monophosphate; HGPRT,
hypoxanthine guanine phosphoribosyltransferase; PNP, purine nucleoside
phosphorylase
In this work, we identified a novel scaffold that targets IMPDH with
low micromolar potency against Mtb. The cellular mechanism of this
compound was demonstrated by selection of resistant mutants that
resulted in amplification of guaB2 gene expression as well as by the
ability of exogenously supplied guanine to rescue their inhibition. We
report the kinetics of enzyme inhibition and were able to show by
structural analyses that the inhibitor bound to the IMP cofactor in the
enzyme active site. Despite the cidality of these IMPDH inhibitors in
vitro, these compound had limited efficacy in vivo and further
quantification of guanine in granulomas from infected human and rabbit
tissues, showed high local concentrations of this nucleotide precursor
limiting the potential of IMPDH as a drug target for Mtb.
RESULTS
Identification and phenotypic characterization of an antitubercular indazole
sulfonamide scaffold
In a screen of 100,000 compounds for inhibitors of growth of Mtb
(manuscript in preparation), an indazole sulfonamide (1) was discovered
with low micromolar potency against the organism ([149]Table 1). This
compound was attractive from a medicinal chemistry perspective based on
its lack of cytotoxicity, acceptable physicochemical properties, high
solubility, synthetic feasibility ([150]Supplementary method
[151]Scheme 1) and acceptable in vitro
absorption-distribution-metabolism values ([152]Table S1).
Table 1.
Indazole sulfonamides in this work and their anti-tubercular potencies
Compound ID Structure MIC (µM)[153]^a
1 graphic file with name nihms967272t1.jpg 2
2 graphic file with name nihms967272t2.jpg 2
3 graphic file with name nihms967272t3.jpg >50
4 graphic file with name nihms967272t4.jpg 6.2
5 graphic file with name nihms967272t5.jpg 9.5
6 graphic file with name nihms967272t6.jpg 0.09
7 graphic file with name nihms967272t7.jpg >50
[154]Open in a new tab
^a
MIC values for compounds 1–4 are for Mtb H37Rv and for compounds 5–7
are for M. bovis BCG. MIC for compounds 6 and 6 against Mtb H37Rv were
0.2 and >50 µM, respectively.
Scheme 1.
[155]Scheme 1
[156]Open in a new tab
Reagents and conditions: a) 5- or 6-indazole (1 eq.), sulfonyl chloride
(1 eq.), 3, 5-lutidine (4 eq.), DCM (2 mL/mmol), 16 h, rt. b) Indazole
(1 eq.), sulfonyl chloride (1.2 eq.), pyridine (0.67 mL/mmol), 16 h, 80
°C
A literature search showed no precedent to guide an understanding of
its possible mechanism of action. Since many anti-tubercular drugs in
clinical use, and in the drug development pipeline, target aspects of
cell wall biosynthesis, we first set out to evaluate this. We had
previously developed an assay that measures the extent of upregulation
of the promoter of the iniBAC gene cluster, known to be induced by
inhibitors of cell wall biosynthesis ^[157]15, by generating a reporter
construct where this promoter drives expression of firefly luciferase
^[158]16. Drugs such as isoniazid, ethionamide, SQ109 and ethambutol
that inhibit cell wall mycolate or arabinan biosynthesis, increase
luciferase expression in the first 48 hours of exposure ^[159]16.
Initial profiling of compound 1 indicated that this promoter was not
upregulated at these early time points, but was upregulated after 72
hours of drug exposure suggesting a possible downstream effect on cell
wall synthesis ([160]Fig. 2A). To further confirm an effect on cell
wall integrity, we measured the kinetics of extracellular release of an
intracellularly expressed green fluorescent protein ^[161]17 during
compound exposure. GFP was released from bacteria indicating that the
compound was lytic and these effects were observed to be subsequent to
the upregulation of the iniBAC promoter ([162]Fig. 2B).
Figure 2. Indazole sulfonamides induce slow lysis of whole cells of Mtb by a
mechanism involving perturbation of purine metabolite pools.
[163]Figure 2
[164]Open in a new tab
A. Effects of compounds 1 (C1), 2 (C2), isoniazid and moxifloxacin at
their MIC concentrations on expression of the cell wall responsive
iniBAC promoter as measured using the pini-luc strain. B. C1 results in
release of cytosolic proteins as measured by GFP-based fluorescence in
culture supernatant during exposure to the compound. C. Scanning (rows
1 and 2) and transmission (row 3) electron microscopy of untreated or
C1 exposed cells at 1× (column 2) or 10X MIC values. D. The indazole
sulfonamides do not inhibit peptidoglycan biosynthesis at 1X and 10X
MIC concentrations as measured by radiolabeling of the macromolecule
using ^14C-N-acetyl-D-glucosamine. The positive controls
meropenem/clavulanate (MCA) and tunicamycin were used at 10X MIC
values.
To understand better the effects of the indazole sulfonamide on the
integrity of the cell wall ultrastructural architecture, we analyzed
exposed cells by scanning electron microscopy at time points
corresponding to early stages of cell lysis. Compound 1 caused the same
polar swelling and cellular elongation observed with β-lactams
^[165]17([166]Fig. 2C). Transmission electron microscopy revealed a
similar accumulation of electron-opaque density in the periplasmic
region separating the plasma membrane from the outer cell wall layers
as had been observed in cells treated with other inhibitors of cell
wall biosynthesis ^[167]18([168]Fig. 2C). These results suggested that
an aspect of cell wall biosynthesis was inhibited, possibly
peptidoglycan. However, a macromolecular incorporation assay using
radiolabeled N-acetylglucosamine to quantitate effects on peptidoglycan
biosynthesis, revealed that compound 1 did not affect incorporation of
this precursor into this macromolecule ([169]Fig. 2D).
To investigate the effects of the indazole sulfonamide on the
metabolism of Mtb, we exposed monolayers of cells to increasing
concentrations of both an active analog (1) as well as a poorly active
sulfonate derivative (3) ([170]Table 1) of this compound. Unbiased
analysis of the corresponding metabolic pathways indicated that purine
metabolism was the most affected pathway with 9 out of 92 total
enzymatic reactions in this pathway showing perturbations in metabolite
pools ([171]Tables 2 and [172]S2). [173]Table 2 shows the detailed
results of the pathway analysis obtained using the pathway tool
MetaboAnalyst 3.0^[174]19. Metabolomic analysis of the purine
biosynthetic pathway showed that at equimolar concentrations, the
active analog (1) resulted in profiles distinct from that of the poorly
active derivative (3) with marked accumulation of inosine-based
nucleotides as well as several pyrimidine and adenine-based nucleotides
([175]Fig. 2A). The concomitant decrease in xanthosine monophosphate
led us to examine the dose dependent effects of compound 1 on
intracellular inosine monophosphate (IMP), xanthosine monophosphate
(XMP) and GMP which showed that the accumulation of IMP was inversely
associated with concentrations in XMP and GMP pools ([176]Fig. 3B).
Table 2.
Pathway enrichment analysis of compound 1 on the Mtb metabolome
Pathway Hits[177]^a Total[178]^b Raw p value[179]^c Holm
adjusted p
value[180]^d FDR[181]^e
Purine metabolism 9 92 3.9E-08 3.16E-06 3.16E-06
Lysine biosynthesis 4 32 0.00016 0.012 0.006
Arginine/proline metabolism 4 77 0.0046 0.356 0.122
Pyrimidine metabolism 3 60 0.016 1 0.312
Alanine, aspartate and glutamate metabolism 2 24 0.019 1 0.312
Aminosugar and nucleotide sugar metabolism 3 88 0.044 1 0.587
[182]Open in a new tab
^a
the actual number of matched compounds within the associated pathway;
^b
the total number of compounds in the pathway; the column labeled ‘Hits’
indicates;
^c
original/uncorrected p value calculated from the enrichment analysis;
^d
the p value adjusted by Holm-Bonferroni method;
^e
the FDR is the p value adjusted using False Discovery Rate.
Figure 3. Indazole sulfonamides induce accumulation of IMP and reduction of
XMP and GMP levels.
[183]Figure 3
[184]Open in a new tab
A. Heatmap of intracellular metabolite concentrations in purine
metabolism as a function of concentration of active (AC) and inactive
(IN) analogs 1 and 3, respectively. Cells were treated with analogs at
equimolar concentrations corresponding to 0, 0.5, 1, 5 and 10X MIC
values of C1. B. C1 results in dose dependent intrabacterial (IB)
accumulation of IMP with concomitant decreases in XMP and GMP
metabolite pools likely as a result of IMPDH inhibition.
Mutants with acquired resistance suggest guaB2 overexpression
We selected for mutants that were spontaneously resistant to 10-fold
MIC levels of compound 1 and compound 2 ([185]Table 1) on solid media
and found that these appeared at a frequency of 1 × 10^−9. These
mutants were subsequently confirmed to be 8- and greater than 32-fold
resistant to the sulfonamide scaffold ([186]Table 3). Whole genome
sequencing of 2 mutants that were obtained revealed that, while both
had single nucleotide polymorphisms (SNPs) in nadD (encoding the
nicotinate-nucleotide adenylyltransferase), one mutant had a SNP in the
likely promoter region of guaB2 (Rv3411c) whereas the other mutant had
an approximately 20-fold duplication of a 50kb genomic region spanning
Rv3371 to Rv3411c ([187]Fig. 4A, [188]Table 3).
Table 3.
Mutation of indazole sulfonamide resistant mutants
ID Deletions Duplications SNPs MIC (fold)
SR2.1 plcD ~20-fold duplication of Rv3371-Rv3411c nadD:G180A 32~64
Rv1787- Rv1790
SR2.2 None None nadD:V14I, nrdZ:C67F 8
G>A :−49 bp of Rv3411c
[189]Open in a new tab
Figure 4. Resistance to the indazole sulfonamides develops through an unusual
gene amplification of a 40 gene region that includes IMPDH.
[190]Figure 4
[191]Open in a new tab
A. Density of reads across the genome as measured by Illumina-based
sequencing. B. Quantitative PCR analysis of Rv3392 inside the amplified
region as compared to mviN (control outside of amplified region) in the
SR2.1 sulfonamide resistant mutant as compared to parental strain
showing >10 amplification. C. Guanine, but not guanosine or inosine,
rescue growth inhibition by C1.
Using the available X-ray crystal structure of NadD in complex with
NADP (PDB ID: 4YBR), we analyzed the effects of the predicted NadD
amino acid substitutions on the protein structure and function using a
previously established pipeline ^[192]20. V14 is located at the dimer
interface between the two NadD protomers, making local hydrophobic
intra and inter molecular interactions. Mutation to isoleucine is not
predicted by SDM ^[193]21 and DUET ^[194]22 to affect the stability of
the protomer. There is sufficient space at the interface to accommodate
the isoleucine and maintain the hydrophobic interactions, and
accordingly this mutation is not predicted by mCSM-PPI ^[195]23 to
destabilize the homodimer. V14 is located 8 Å away from the NAD ligand,
and mutation to isoleucine is predicted by mCSM-lig ^[196]24 to mildly
decrease binding affinity. G180A is a surface mutation of a negative
phi glycine on an alpha helix of NadD, which SDM and DUET do not
predict will destabilize the protomer, and mCSM-PPI predicts this
change will have minimal effect on formation of the homodimer. The
mutation is located 16.6 Å away from the NAD binding site, and is
predicted by mCSM-lig to have minimal effect on the affinity for NAD.
This suggested that these mutations were unlikely to play a direct role
in the resistance observed to these compounds.
Quantitative PCR confirmed the 20-fold amplification of the Rv3371 to
Rv3411 spanning region originally observed in the whole genome
sequencing ([197]Fig. 4B) and quantitative RT-PCR analysis confirmed
that the SNP in the promoter of Rv3411c caused upregulation of guaB2
transcript expression ([198]Fig. S1). Quantitative PCR of genomic DNA
of the mutant compared to the parental strain showed that this gene was
amplified 14-fold in the genome of the resistant mutant (results not
shown). We reasoned that, if the mechanism of growth inhibition
involved the essential Mtb inosine monophosphate dehydrogenase (IMPDH)
encoded by guaB2 that catalyzes NAD^+-dependent oxidation of IMP to
xanthosine monophosphate (XMP) in the de novo biosynthetic pathway for
guanosine nucleotides, growth inhibition would be overcome by guanine
supplementation ^[199]25. Guanine rescue would only be possible in the
presence of a functional purine salvage pathway in the cell conferred
by the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) encoded
by the hpt gene that phosphoribosylates hypoxanthine or guanine to
replenish purine nucleotide pools ^[200]26. Indeed, concentrations of
guanine above 100 µM rescued cells from the effects of 1 and 2
([201]Fig. 4C, and [202]Table S3) showing that the salvage pathway
utilizing the activity of HGPRT could overcome metabolic blockage of
IMPDH. In contrast, guanosine and the IMPDH substrate inosine could not
rescue growth likely due to lack of uptake mechanisms of these
nucleosides ([203]Fig. 4C). As expected, no other nucleobases could
rescue Mtb from indazole sulfonamide (results not shown).
Indazole sulfonamides are uncompetitive inhibitors of IMPDH
We next confirmed the ability of this compound to inhibit Mtb IMPDH in
vitro. Recombinant Mtb IMPDH was expressed as a truncated isoform of
the catalytically active core after deleting both cystathione
β-synthase (CBS) domains ^[204]27; with no alteration on its steady
state kinetic constants when compared to native Mtb IMPDH^[205]12.
Compound 1 displayed an IC[50] of 0.38 ± 0.02 µM (r^2 0.99) in the
enzyme assay ([206]Fig. 5A). Moreover, comparison of in vitro enzyme
inhibition values to potency of analogs (manuscript in preparation)
against whole cells showed remarkable correlation (R^2 of 0.8)
([207]Fig. 5B).
Figure 5. Indazole sulfonamide inhibits Mtb IMPDH.
[208]Figure 5
[209]Open in a new tab
A. Kinetics of Mtb IMPDH inhibition. B. Correlation between Mtb IMPDH
inhibition and potency against Mtb cells as measured by MIC. C.
Identification of IMPDH as a target by chemoproteomics. C5 was
covalently immobilized to NHS-activated sepharose beads at the primary
amine. The beads were incubated with M. bovis BCG extract either in the
presence of vehicle (DMSO), or in the presence of C6 (active) or the
inactive analog C7. Relative quantification of all proteins captured on
the beads was performed by isobaric peptide tagging and LC-MS/MS. A
single protein, IMPDH (BCG_3481c, GuaB2), showed specific and selective
binding as indicated by loss of binding in the presence of excess C6,
but not C7. D. Affinity capturing on beads in the presence of different
concentrations of “free” compounds allowed the determination of an
IC[50] value of 0.8 µM and an apparent dissociation constant (K[d]^app)
of C6 for IMPDH, whereas C7 shows only very weak binding. Data shown
are the results of two replicate experiments.
To further confirm target engagement in the context of the cellular
environment, a chemoproteomic approach was used to identify potential
binding partners from the mycobacterial proteome ^[210]28. This
strategy is based on the immobilization of chemical analogs of the
active compound to beads, which are subsequently incubated with
bacterial extract. Proteins captured by the beads were identified after
tryptic digestion and liquid-chromatography-tandem mass spectrometry
(LC-MS/MS). We prepared indazole sulfonamide analogues which
derivatized the active pharmacophore with different types of linkers
and a primary amino group, allowing covalent attachment to Sepharose
beads. Compound 5 retained anti-bactericidal activity ([211]Table 1)
suggesting that the derivatization with the linker moiety did not
interfere with target binding. The derivatized beads were incubated
with Mycobacterium bovis BCG extract and proteins captured by the beads
were digested with trypsin, labeled with isobaric mass tags (TMT
10plex), and quantitatively identified by LC-MS/MS. In order to
distinguish true targets from nonspecific background binding, aliquots
of the bacterial extracts were incubated prior to the pulldown step
with either the antibacterial compound 6, or with the structurally
related inactive compound 7 ([212]Table 1). The active compound, but
not the inactive analog, is expected to bind to the target protein(s)
in the lysate and thus reduce the binding of these proteins to the
beads. IMPDH (BCG_3481c, GuaB2) was the only protein in our experiments
that exhibited this behavior suggesting that the active compound is a
selective IMPDH inhibitor ([213]Fig. 5c). The inactive compound 7
showed only partial competition of bead-binding with IMPDH even at the
high concentration of 40 µM ([214]Fig. 5d). GuaB1 and GuaB3 were
captured by the beads to some degree, but this binding was not affected
by excess compound 6, suggesting that they are not targets of compound
6. In order to estimate inhibitor potency, we performed the pulldown
step in the presence of different concentrations of “free” compounds,
which allowed the determination of an IC[50] value of 0.8 µM for IMPDH.
The IC[50] value represents a measure of target affinity, but is
affected by the affinity of the target for the bead-immobilized ligand.
The latter effect can be deduced by measuring the depletion of the
target protein by the beads ^[215]29. The apparent dissociation
constant (K[d]^app) of compound 6 for IMPDH was determined to be 0.7
µM. In order to exclude potential adverse effects mediated by
modulation of host (human) targets, we employed the same strategy to
evaluate binding to proteins in extracts from human material (K562
erythroleukemia cells, HEK embryonic kidney cells, and placenta
tissue). Notably, the human orthologs IMPDH1 and IMPDH2 were captured
by the indazole sulfonamide beads, but were not affected by
preincubation with excess “free” compound 6, indicating a high degree
of selectivity for the bacterial over the human enzyme.
The mode of enzyme inhibition is critical in evaluating the potential
of a compound as a growth inhibitor in vivo since the extent of
inhibition of the reaction can be determined by substrate and/or
product concentrations depending on inhibitor kinetics. Kinetic
evaluation of compound 1 showed the mode of inhibition was
uncompetitive with IMP and NAD^+ ([216]Fig. 6) with a Ki of 0.220 µM.
Uncompetitive inhibitors are appealing in that enzyme inhibition could
lead to build-up of substrate further driving enzyme inhibition. In
addition, the IC[50] of compound 1 against the human IMPDH was found to
be 15 µM ([217]Fig. S2) showing a selectivity index of approximately 40
for the Mtb IMPDH.
Figure 6. The indazole sulfonamide is an uncompetitive inhibitor of Mtb
IMPDH.
[218]Figure 6
[219]Open in a new tab
The mechanism of inhibition of compound 1. The Lineweaver-Burk plots
(upper) were generated to display the type of inhibition, which is
uncompetitive for both IMP and NAD^+.
X-ray structure of indazole sulfonamide, compound 1 and IMPDH
The Mycobacterium thermoresistible (Mth) IMPDH protein which shares 85%
amino acid identity with the Mtb IMPDH, including a 100% conservation
of residues in the active site, was chosen for structural studies since
it gave higher protein expression yields than the Mtb homolog. Mth
IMPDH ΔCBS crystallized in the I4 space group and diffracted to sub 2 Å
resolution. One protomer was present in the asymmetric unit, with the
biological tetramer observable through operation of 222 symmetry of the
crystal lattice ([220]Fig. 7A). In the X-ray crystal structures of
compound 1 with Mth IMPDH, clear electron density for the compound was
observed within the NAD binding pocket of IMPDH in the 2F[0]-F[c]
difference map (σ = 3.0), stacking with IMP ([221]Fig. 7B, C). It is
worth noting that the majority of interactions mediated by compound 1
within the crystal structure are through extensive pi interactions
between the indazole group and the hypoxanthine group of IMP ([222]Fig.
7E; [223]Fig. S3) consistent with the uncompetitive binding mode
suggested by the enzyme kinetics. The indazole is able to make further
pi interactions with A285 (A269 in the Mth structure), and polar
interactions with G334 and T343 (G318 and T327 in the Mth structure
respectively). The pyrazole makes further pi interactions to A269, in
addition to some proximal hydrophobic interactions to E458 (E442 in the
Mth structure). Additional polar interactions are mediated by the
sulfonyl group to G425 (G409 in the Mth structure).
Figure 7. The X-ray crystal structure of Compound 1 and Compound 6 bound to
IMPDH.
[224]Figure 7
[225]Open in a new tab
A. The IMPDH tetramer (cyan ribbon, with a representative protomer
shown in grey) is shown bound to IMP (blue) and Compound 1 (C1)
(orange) is shown. B. The structural alignment of the IMPDH crystal
structures of C1 (orange) and Compound 6 (C6)(Magenta), showing the
inhibitors are orientated identically in the NAD+ binding pocket. C, D.
2F0-Fc difference maps (σ = 3.0) showing clearly visible electron
density for C1 (C) and C6 (D) in the NAD+ binding site. E. Interactions
made by C1 (E) (orange) in the X-ray crystal structure of the complex
of IMPDH (grey; and adjacent protomer in cyan) with IMP (blue). Residue
numbering is of the corresponding residues in Mtb. Pi interactions are
shown in green, hydrogen bonds in red, polar interactions in orange and
proximal hydrophobic interactions in grey. The solid lines are covalent
bonds, and the dashed lines are non-covalent interactions.
From crystals soaked with compound 6, the 2F[0]-F[c] difference map (σ
= 3.0) revealed strong density for the inhibitor ([226]Fig. 7D). The
structure of compound 6 showed the compound bound in a near identical
manner to compound 1 ([227]Fig. S3), with the indazole sulfonamide
taking advantage of the same extensive interactions to IMP, in addition
to a few interactions with neighboring residues in the binding pocket
(A285, G334, T343, G425 and E458). The fluorophenyl acetamide extension
of Compound 6 is able to make polar interactions with T284, A 285 and
H286 (T268, A269 and H270 in the Mth structure, respectively), and the
benzene group making proximal hydrophobic interactions to H286, N289
and V292 (H270, N273, and V276 in the Mth structure, respectively).
Notably, however, the fluorine is in good orientation to make a 3.2 Å
hydrogen bond with the side-chain of N289, helping to lock in the
orientation of the compound.
Indazole sulfonamides are growth dependent inhibitors of Mtb
Having confirmed the on-target inhibition of IMPDH both in vitro and in
the context of cellular metabolism, we explored the physiological
consequences of IMPDH inhibition on Mtb survival. Exposure of Mtb to
compound 1 and 2 showed that IMPDH inhibition resulted in slow
bacterial death at high concentrations whereas MIC levels of compounds
resulted only in bacterial growth inhibition ([228]Fig. 8A). The
kinetics of cidality recapitulated the late upregulation of the cell
wall responsive iniBAC promoter and bacterial lysis observed during
treatment of Mtb with these compounds ([229]Fig. 2A). We confirmed that
these compounds exerted a growth inhibitory effect in macrophages
([230]Fig. 8B), although high concentrations were required to effect
bacterial stasis. The vulnerability of IMPDH during non-replicative
bacterial persistence in vitro was determined by treating starved or
anaerobically adapted Mtb with compound 1 or 2 which showed that
exposures as long as 3 weeks at 100-fold MIC levels of compound did not
significantly affect bacterial survival ([231]Fig. 8C, D), arguing
against the vulnerability of this target during non-replicating
persistence. These results suggest that IMPDH inhibitors were only
effective against replicating Mtb cells.
Figure 8. The indazole sulfonamide is cidal for replicating cells but lacks
activity in non-replicating cells and in murine infection.
[232]Figure 8
[233]Open in a new tab
A. Logarithmically growing Mtb was exposed for 7 days to compounds 1
(C1) and 2 (C2) at 1, 20 and 50X MIC values. Control cells were exposed
to 10X MIC concentrations of Rifampicin (RIF). B. C1 lacks cidality
against Mtb during growth in macrophages. Mtb-infected J774 macrophages
were exposed to C1 at 10, 50 and 100X MIC concentrations for 7 days
prior to CFU enumeration. RIF and DMSO were used as positive and
negative controls, respectively. C. C1 is inactive against
anaerobically persisting cells. Anaerobically adapted cells were
exposed up to three weeks to C1 at 20 and 100X MIC values prior to CFU
enumeration. Metronidazole at 100 µM was used as positive control.
Isoniazid (100µM) and DMSO were used as negative controls. D. C 1 lacks
efficacy against starved non-replicating Mtb. Two-week starved Mtb
cultures were exposed up to 3 weeks to C1 and RIF at 10X MIC values
prior to CFU enumeration. C 1 lacks efficacy in acute (e) and chronic
(f) stages of murine infection as measured by CFU analysis of lung
tissues. Mice were dosed at 10, 30 and 100mg/kg of C1 and C2 with
vehicle and 10 mg/kg RIF treated mice serving as negative and positive
controls, respectively.
To establish the vulnerability of IMPDH during host pathogenesis, we
first sought to verify the efficacy of these compounds in an animal
model that supports Mtb replication. Mtb replicates in lungs of both
acute as well as chronically infected C57BL/6 mice, in chronic stages
of infection replication being balanced by bacterial death^[234]30. Mtb
infected C57BL/6 were treated with 10 to 100 mg/kg of 1 and 2 with
dosing initiated 2 weeks post infection as well as in 7-week infected
mice where a chronic infection had been established. Our results showed
a surprising lack of efficacy in both stages of infection as observed
by a lack of effect on bacterial organ burdens compared to treatment
controls ([235]Fig. 8 E, F).
Factors that contribute to lack of in vivo efficacy
The lack of in vivo efficacy of the indazole sulfonamide contrasted
with its in vitro efficacy and led us to explore the factors that
contributed to this. The finding that guanine could rescue the cidality
in vitro ([236]Fig. 4C, [237]Table S3) could suggest that the pathogen
employed scavenging mechanisms for host derived guanine similarly to
the scavenging of host derived nicotinamide in NAD salvage observed
during growth in macrophages^[238]31. Labeling of cells growing
axenically in vitro or released after growth in macrophages^[239]31
with radiolabeled guanine indicated that guanine uptake mechanisms were
downregulated during parasitism of the host ([240]Fig. 9A) as compared
to rapidly in vitro replicating cells, arguing against increased
salvage of host purines contributing to the discrepancy between in
vitro and in vivo efficacy.
Figure 9. Failure to achieve murine efficacy is likely due to sub-optimal
exposure, P-GP mediated efflux and high lesion guanine levels.
[241]Figure 9
[242]Open in a new tab
A. Mtb does not upregulate guanine uptake during host pathogenesis. Mtb
growing in macrophages was compared to logarithmically growing cells
for their radiolabeling by ^14C-guanine. B. Extended exposure of Mtb to
compound 1 (C1) at 10- and 20X MIC concentrations is required to effect
cidality. Logarithmically growing Mtb was exposed for 7 days to
compounds for daily exposure periods of 3h, 6h or 24h followed by
compound removal. C. Pharmacokinetics of C1 and C2. D. Measurement of
permeability and efflux of C1 and C2 in a Madin Darby canine kidney
cell permeability assay. E. Guanine concentrations in microscopically
unaffected tissue and in lesions in resected lung tissue from two
tuberculosis patients.
We next sought to understand whether inhibitor concentrations at the
site of infection could have played a role in lack of in vivo efficacy.
We analyzed the compound exposure required to effect bacterial killing
in vitro and during infection of host macrophages by daily addition and
removal of drug after either 3 or 6 hours of drug exposure compared to
constant exposure (24 h). These studies suggested that continuous
exposure at 10-fold MIC values was required to exert a cidal effect in
vitro ([243]Fig. 9B) and stasis in macrophages ([244]Fig. S4). Analysis
of the guanine concentration required to rescue growth inhibition
demonstrated that concentrations of 10 µM guanine showed partial rescue
of growth in indazole sulfonamide concentration dependent manner
whereas 100 µM could fully overcome all growth inhibition ([245]Fig.
S5).
Pharmacokinetic analysis of blood concentrations of the two indazole
sulfonamides (compounds 1 and 2) used for treatment of infected mice in
[246]Fig. 8 E & F showed that, although the highest dose achieved a
maximal serum concentration more than 10-fold higher than MIC values
and an Area-Under-the-Curve (AUC) value more than 30-fold higher than
MIC values ([247]Fig. 9C), the compound was well below the effective
cidal concentration after 8 hours of dosing. The AUC following oral PK
showed that for compound 1 there was proportional increase as the dose
was escalated whilst for compound 2, there was a proportional increase
between 5 and 30mg/kg but not between 30 and 100 mg/kg as the exposure
was moderate when compared to each other ([248]Table S11). Analysis of
drug concentrations for compound 1 in bronchoalveolar lavage analyses
similarly showed that despite drug reaching high concentrations in the
epithelial lining fluid, concentrations at 4 hours after dosing were
below the efficacious concentration and a reflection of the free drug
concentration in blood ([249]Tables S11 and S12). Moreover, analysis of
permeability and efflux of this scaffold in a Madin Darby canine kidney
cell permeability assay indicated that this scaffold was a likely
P-glycoprotein (P-gp) substrate ([250]Fig. 9D) which could additionally
have contributed to low microenvironmental concentrations of these
compounds in the mouse lung.
Our result suggested that efficacy in the mouse model could be improved
by developing a compound with better serum exposure and lower P-gp
efflux. However, the finding that guanine concentrations determine the
efficacy of an IMPDH inhibitor in vitro led us to explore guanine
concentrations in lung tissue. Mtb-infected rabbits develop granulomas
with many of the defining characteristics of human granulomas ^[251]32.
Dissecting caseous lesions from Mtb-infected rabbits allowed
quantitation of free guanine levels in rabbit caseum directly and
revealed guanine concentrations in the range of 0.2–0.5 mM in both
uninvolved lung tissue and in lesions in rabbits that is greater than
the 0.1 mM guanine required for rescue ([252]Fig. S6). Similarly,
curated samples of human tissue from patients with refractory
multidrug-resistant tuberculosis who had undergone surgical resection
for the treatment of their disease were analyzed for guanine content.
Granuloma and cavity caseum had between 0.4 and 0.8 mM guanine and
visually uninvolved lung tissue from the same two patients had 2 to 3
times more guanine than present in the lesion tissue ([253]Fig. 9E). In
contrast, J774 cells had guanine concentrations (43 µM) in the range of
that reported for subpopulations of human cells^[254]33 whereas the
guanine concentrations in mouse tissues ranged from 7 – 20 µM
([255]Table S13, Fig. S7), concentrations that could partially rescue
growth inhibition ([256]Fig. S5).
DISCUSSION
The iniBAC promoter is a reporter of cell wall insult that rapidly
responds to broad classes of cell wall inhibitors within 24 hr
^[257]15, [258]16. In the case of the indazole sulfonamides we
initially classified them as not cell wall active based on the absence
of this response but noted with interest that these compounds induced
cell lysis and resulted in a delayed firing of the iniBAC promoter. We
had seen a similar pattern of in vitro behavior previously with
meropenem, a β-lactam of the carbapenem family. In this case this
phenotype was accompanied by a unique polar swelling visualized by
electron microscopy ^[259]17. The indazole sulfonamides showed a
similar swelling at the cell poles but did not exhibit any direct
effect on incorporation of ^14C-N-acetylglucosamine, a precursor to
peptidoglycan, suggesting that they did not exert a direct effect on
peptidoglycan biosynthesis despite these similarities to β-lactams. The
metabolic profiles resulting from treatment of Mtb cells with the
indazole sulfonamides strongly suggested an effect on purine nucleotide
pools suggesting that the cell wall effects were downstream
consequences of purine nucleotide pool perturbations.
Mutants resistant to the indazole sulfonamides proved remarkably
difficult to select and only occurred at very low frequency. Sequencing
of these mutants at first revealed no helpful SNPs to suggest the
actual target, instead we observed a 2–20 fold tandem duplication of a
nearly 50 kb pair region of the chromosome spanning 40 genes from
Rv3371 to Rv3411c. Remarkably, large scale repeats of genes in this
region of the chromosome have been reported in the Pasteur strain of
BCG, the Beijing strain of Mtb, and more recently in several other
modern TB lineages ^[260]34–[261]36. The only resistant mutant we
obtained that did not show this gene amplification harbored a SNP just
upstream of the last gene in the amplified region, guaB2. The finding
that guanine supplementation rescued Mtb from growth inhibition by this
scaffold confirmed the notion that the mechanism of action was related
to inhibition of IMPDH.
The connection between IMPDH inhibition and the observed effects on
cell envelope integrity was unexpected but three facts are worth
considering. The observed cell lysis occurs very slowly and only after
about five days, with the iniBAC promoter assay becoming positive
slightly earlier at about 3 days. The central role of guanine
nucleotides in protein synthesis and biosynthesis of the essential
flavin cofactor required for a plethora of reactions including
UDP-galactopyranose mutase, could additionally explain the downstream
effects on cell wall integrity.
The very low observed frequency of resistance suggested the possibility
that there were multiple cellular targets for the indazole
sulfonamides. Two experiments suggest that this is not the case. First,
across a structurally diverse set of analogs of this series with MICs
ranging from 100 nM to 100 µM the IC[50] values against IMPDH showed a
strong correlation. Second, attachment of an indazole sulfonamide
analog to beads specifically pulled IMPDH out of whole cell lysates and
this could be competed with an active analog but not with a closely
related inactive analog ([262]Fig 5C). A more likely explanation for
the low frequency of resistance appeared when we solved the X-Ray
crystal structure of our lead compound in complex with the IMPDH from
M. thermoresistible. Full-length Mth IMPDH (GuaB2) has 85% sequence
identity with Mtb IMPDH and is 100% conserved in the active site, and
was chosen for further crystallographic studies due to its higher
expression levels and ready crystallization in a soakable crystal form.
This structure showed that the inhibitor bound primarily to the
substrate IMP at the active site and made relatively few contacts with
the protein. The relative lack of direct interaction with the protein
itself suggests that mutation of the target is unlikely to give
resistance, leaving gene amplification as the only viable route for
acquisition of resistance. This gene amplification may occur at an even
lower rate in vivo since amplification of this region in vitro leads to
mutants with impaired virulence^[263]37. The two non-active site
mutations observed in nadD, encoding the nicotinate mononucleotide
adenylyltransferase involved in NAD biosynthesis, in our resistant
mutants are intriguing. We have confirmed that our compound even at 100
µM does not inhibit MtNadD further corroborating the notion that these
non-active site mutations are not related to the mechanism of action of
the compound.
Consistent with their induction of cell lysis, the indazole
sulfonamides were cidal to actively replicating cells over a one week
incubation. And consistent with the anabolic role of the products of
this enzyme, this series showed no significant cidal activity against
cells in which replication had been arrested either by hypoxia. We
therefore expected to see an effect of these compounds in acute murine
models of disease and for that activity to be significantly curtailed
in chronic disease models where replication is slow. Surprisingly we
saw no activity of these agents in either model despite what appeared
to be suitable exposures. We considered several explanations for the
lack of in vivo activity including that the bacteria might have
upregulated their ability to scavenge guanine derived from the host and
therefore be less susceptible to IMPDH inhibitors. Uptake of
[^14C]-guanine, however, was not upregulated in Mtb cells released from
macrophages compared to those in in vitro culture. Cidal activity was
very concentration dependent and at the actual MIC, the compounds were
sufficient to block replication but killing was only apparent above 10X
MIC concentrations. To mimic the exposures seen in vivo, we next
assessed what duration of exposure was required to achieve cidal
activity and did daily pulses of exposure for 3, 6 or 24 hours at 20X
MIC and found that even 6h at 20X MIC was insufficient to kill cells.
We also found that these compounds were subject to active efflux by
P-gp so the actual exposure of bacteria within macrophages was likely
considerably lower than what was measured in the serum PK study.
Another potential explanation for the lack of in vivo efficacy was that
host guanine levels were sufficient to rescue the IMPDH inhibitory
effect. To assess this we measured free guanine levels in mouse, rabbit
and human TB lesions and found surprisingly high concentrations of
guanine in normal lung tissue as well as in the caseum of granulomas of
rabbits and humans whereas normal lung tissue of uninfected mice was
10–13 µM. High intracellular concentrations of guanine have previously
been reported in both E. coli^[264]38, [265]39 (~180 µM) and in normal
human cells^[266]33 (70–800 µM). Intracellular guanine concentrations
in J774 macrophages used in our work were measured to be 43 µM, levels
not high enough to fully rescue growth inhibition by the indazole
sulfonamide although high enough to partially alleviate cidality.
Although the mouse lung concentrations do not explain the lack of
efficacy in this animal model, these levels are a concern for IMPDH as
a drug target in humans where salvage of extracellular guanine could
provide a bypass mechanism for decreased flux through the de novo
pathway ([267]Fig. 1). The most recalcitrant bacteria are thought to be
the non-replicating bacilli found within the necrotic core of lesions
where guanine was the lowest but where the metabolic requirement for
guanosine nucleotides is minimal as evidenced by the growth rate
dependence of IMPDH inhibition. In combination, these data suggest that
IMPDH, although essential for Mtb survival in vitro in laboratory
growth media, is relatively invulnerable and has low potential for
achieving treatment-shortening in humans infected with Mtb.
MATERIALS AND METHODS
Animal care and human ethics assurance
Mouse and rabbit studies were carried out in accordance with the Guide
for the Care and Use of Laboratory Animals of the National Institutes
of Health under Animal study protocol numbers LCID 4E and LCID3,
additional rabbit studies were done with approval from the
Institutional Animal Care and Use Committee of the New Jersey Medical
School, Newark, NJ under Rutgers Animal Welfare Assurance Number
A3158-01. For human samples, anonymized lung tissue containing
granulomas were collected from patients with treatment refractory TB
during therapeutic lung resection surgery at National Masan Hospital,
Republic of South Korea. The collection was approved by the hospital’s
institutional review board, an exemption from National Institutes of
Health, Office for Human Research Protections, and with written,
informed consent of the subjects. All regulated procedures on living
animals performed at the University of Dundee were carried out under
the authority of a license issued by the Home Office under the Animals
(Scientific Procedures) Act 1986, as amended in 2012 (and in compliance
with EU Directive EU/2010/63). License applications will have been
approved by the University's Ethical Review Committee (ERC) before
submission to the Home Office. The ERC has a general remit to develop
and oversee policy on all aspects of the use of animals on University
premises and is a sub-committee of the University Court, its highest
governing body.
Strains and media
Mycobacterium tuberculosis H37Rv was used for all experiments except
pini-luc and GFP release assay. The piniBAC- luciferase expressing
strain and a GFP-expressing Mtb/pMSP12 strain were used for pini-luc
and GFP release assay, respectively ^[268]17. Middlebrook 7H9 (Becton
Dickinson) supplemented with ADC [albumin (50 g l^−1)/dextrose (20 g
l^−1)/NaCl (8.1 g l^−1)], 0.2% glycerol] and 0.05% Tween 80 was used
for liquid media and Middlebrook 7H11 (Becton Dickinson) supplemented
with OADC [ADC with 0.06% oleic acid] was used for solid media for in
vitro growth of Mtb. MIC determinations were performed as previously
described ^[269]40.
Evaluation of Indazole Sulfonamide effects on Mtb cell wall synthesis
The pini-luc strain was grown at 1× MIC of each compound at 37°C for 7
days in 96 well plates. Every 24 h, 50 µl of culture was taken and
mixed with 50 ul luciferase assay buffer [50 mM HEPES pH 8.0, 0.4%
Triton X100, D-luciferin (28 mg l-^1), 50 mM DTT]. The mixture was
incubated at 37°C for 30 min and RLU was measured by FlUOstar Optima
(BMG LABTECH). For the GFP release assay, Mtb/pMSP12 strain was
incubated in 7H9/ADC/Tween with 30 µg/ml of kanamycin in roller bottles
at 37°C to exponential phase (OD 0.2) ^[270]17. The culture was split
to 30 ml in 250 ml roller bottles with test compounds added at 1X and
10X MIC and cultured for 14 days. Each day 1 ml of each culture was
centrifuged at 15,000 g for 5 min. Supernatant was dispensed in 100 µl
aliquots (triplicates) in consecutive wells of 96-well black plate. GFP
fluorescence was measured by FLUOstar optima (λ[ex]485 nm and λ[em] 520
nm) and divided by OD[650] of the culture. For macromolecular
incorporation, Mtb was grown to an OD650nm of 0.4 in 200ml and split
into 11 ml aliquots containing test compounds. After 2 hours at 37°C,
220 µl of 0.1 mCi/ml ^14C-N-acetyl-D-glucosamine (NAG, American
Radiolabeled Chemicals, Inc.) was added to each aliquot. After 24 and
48 hours, 2.5 ml of NAG labelled samples were centrifuged at 3,000 g
for 10 min. The pellet was resuspended in 2 ml CHCl3:CH3OH (2:1) and
incubated at 37°C overnight. Harvested pellets were resuspended well in
200 µl of scintillation fluid and CPM was counted by Scintillation
counter (Beckman Coulter LS6500).
Metabolomic Sample Preparation and Analysis
Samples used for metabolomic analysis of indazole sulfonamide compounds
on viable Mtb cells were prepared using our previously published filter
cultured system^[271]41. In short, Mtb was grown to mid-log phase and
then diluted to an OD[600]nm of 0.1. A 1 ml culture was then inoculated
onto 22 mm 0.2 µm PVDF filters (Millipore) using vacuum filtration,
placed on 7H10+ADN plates, and incubated at 37°C. On day 5
post-inoculation, Mtb-laden filters were transferred to plates
containing DMSO vehicle, 0.5X, 1X, 5X or 10X MIC C1 (or equivalent
molar concentrations of its inactive congener) and incubated for 20h,
at which point there was no grossly measurable loss of viability, as
previously described^[272]42. Samples were then metabolically quenched
by plunging Mtb-laden or mock drug-exposed filters in −20°C
acetonitrile:methanol:H[2]O (40:40:20). Metabolically quenched Mtb
removed from these filters in solution were mechanically lysed with 0.1
mm silica beads in a Precellys tissue homogenizer under continuous
cooling at 2°C. Samples were then clarified by centrifugation and the
supernatant filtered through a 0.22 µm filter. Biomass of each sample
was determined by measuring residual protein content using a
colorimetric assay (Pierce BCA Protein Assay) and used to enable
inter-sample normalization of measured metabolite abundances. Each
experiment included three technical replicates for every condition
tested and was performed twice.
Samples used for metabolomic analysis of lung tissue and macrophages
were prepared by mechanical lysis in −20°C acetonitrile:methanol:H2O
(40:40:20) using a Precellys tissue homogenizer under continuous
cooling at 2°C and processed as described above. Biomass of each sample
was determined by weight and used to determine lesional concentrations
of guanine as described below.
Liquid Chromatography-Mass Spectrometry
Metabolites were separated using a Cogent Diamond Hydride Type C column
(Gradient 3) as previously described^[273]43 and then analyzed using an
Agilent 1200 liquid chromatography system coupled to an Agilent High
Resolution Accurate Mass 6220 TOF. This system achieves mass errors of
approximately 5 ppm, mass resolution ranging from 10,000 to 25,000
(over m/z 121–955), and a 5 log10 dynamic range.
Metabolomic datasets were queried by targeted analysis using Agilent
Profinder 8.0 configured to a mass tolerance of <10 ppm. Putative
metabolite identities were assigned based on accurate mass (m/z) and
chromatographic retention time identifiers and confirmed by co-elution
with authentic chemical standards. Metabolite concentrations were
calculated using the method of standard addition with authentic
chemical standards. Metabolite abundances were normalized within
experiments to residual protein biomass as described above. Absent a
validated internal standard to determine absolute recovery rates, the
reported metabolite abundances and concentrations likely represent
underestimations. Lung tissue and macrophage guanine concentrations
were determined by dividing the normalized metabolite abundances by the
volume of lung tissue (assuming a lung tissue density of approximately
1 g/ml) or total cell volume of macrophages (assuming an approximate
cell volume of 2.1 ul/10^6 cells^[274]44).
Metabolic pathway enrichment analysis was carried out using the online
analytical tool MetaboAnalyst 3.0 ([275]www.metaboanalyst.ca)^[276]19.
Enriched pathways were identified by hypergeometeric test based on a
cumulative binomial distribution.
FDR (false discovery rate)-controlling procedures are designed to
control the rate of Type I, or false positive, errors in large
datasets. FDR methods have greater power (sensitivity) than so-called
familywise error rate (FWER) controlling methods such as
Bonferroni-based corrections but provide less stringent control of Type
I errors^[277]45. Holm–Bonferroni method^[278]46 is a FWER controlling
method used to handle the problem of multiple comparisons that is more
powerful than the standard Bonferroni correction.
Generation and characterization of indazole Sulfonamide resistant mutants
To generate mutants against the indazole sulfonamide scaffold, 50 ml of
Mtb was grown to OD[650] 0.2. Harvested cells were resuspended in 500
µl of media and 100 µl of it (10^9 cells) were plated on 7H11/OACD
plates with 5×, 10×, 50× MIC 1 and 2. They were incubated at 37C for 4
weeks. After 4 weeks, the 2 mutants on 10× MIC compound 2 plates were
inoculated in 7H9/ADC/Tween media. Genomic DNA of mutants was isolated
by CTAB method ^[279]15. Whole genome sequencing was performed and
analyzed as described ^[280]47. To confirm the large duplication of
SR2.1, qPCR were performed with primer sets within the duplication
region (Rv3392) and out of the region (Rv3910). These DNA fragments
were amplified by 25 cycles of PCR with 0.1, 0.2, 0.4, and 1 ng of
genomic DNA of SR2.1 and parental strain. The amount of amplified PCR
product was compared on agarose gels. Quantitative PCR was further used
to confirm amplification of guaB2 in the genome. For this, quantitative
PCR was performed by real-time PCR with SYBR green. One nanogram of
genomic DNA from parental strain and SR2.1 were used for each reaction.
Data was normalized with 16S rRNA gene. Relative gene quantification
was calculated in REST-382©-version1^[281]48. The intergenic sequence
of wild type and mutated guaB2 were used to replace the hsp promoter of
pMV306hsp (Addgene plasmid # 26155), respectively^[282]49. The original
plasmid contains luciferase driven by the hsp promoter. They were
electroporated into M. smegmatis mc^2155. Luciferase activity was
measured as describe above in pini-luc assay. Guanine rescue test was
performed by addition of the supplements to the medium used in the MIC
determination using a final concentration of 100 µM guanine, guanosine,
inosine, xanthine and hypoxanthine.
Efficacy and validating inhibition of Indazole sulfonamide scaffold against
Mtb in vitro and in vivo
In vitro efficacy was performed in aerobic, anaerobic, and starvation
condition. For aerobic conditions logarithmically growing Mtb (OD[650]
0.2) was diluted 1,000 fold in 1 ml of 7H9 media and exposed to 1×,
20×, and 50 × MIC of compound 1 and compound 2 for up to 7days in
duplicates. After 4 days and 7 days of treatment appropriate cell
dilutions were plated on 7H11/OADC plates for CFU enumeration. For
anaerobic conditions, Mtb was cultured in the self-generated
oxygen-depletion model as previously described ^[283]31. One milliliter
of anaerobic Mtb culture was exposed up to 3 weeks to 20X and 100X MIC
compound 1 and compound 2. For the starvation condition,
logarithmically growing 5 ml of Mtb (OD[650] 0.2) culture was washed
with PBST (phosphate buffered saline with 0.05% tyloxapol) 3 times and
incubated in PBST for 2 weeks. Two weeks starved Mtb culture was
aliquot to 1 ml and exposed up to 3 weeks to compound 1 and 2.
Dilutions were plated on 7H11/OADC plates on a weekly basis.
For the ex vivo efficacy test, J774 cells (5× 10^4 cells/well) were
seeded in flat-bottom 96 well plates (Corning incorporated) in DMEM
GlutaMAX (Gibco) supplemented with 10% fetal bovine serum, 20 mM HEPES
+ 0.5 mM sodium pyruvate and infected with Mtb (MOI 1:1) for 24 hours.
Subsequently, cells were washed with PBS (pH 7.4) twice and exposed to
test compounds in the above growth medium. Cells were incubated at
37°C, 95% humidity, 5% CO[2] incubator for 7 days. Media was changed
after 4 days. After 7 days incubation 0.1% SDS was added in each well
to ensure macrophage lysis. After 5 minutes, lysate was rapidly mixed
to shear eukaryotic DNA and diluted in 7H9/ADC and plated on 7H11/OADC
plates.
For evaluation of in vivo efficacy, C57BL/6 mice were infected by the
aerosol route as previously described ^[284]50. After 14 days, groups
of 10 mice were dosed with 1 and 2 given by oral gavage at 10 mg/kg, 30
mg/kg, or 100 mg/kg. Control groups were dosed with vehicle control (1%
carboxymethyl cellulose) or 10mg/kg Rifampicin. After 2 and 4 weeks of
treatment, groups of 5 mice were euthanized and appropriate dilutions
in 7H9/ADC/Tween of organ homogenates plated on 7H11/OADC plates for
CFU enumeration. Similarly, mice were treated by daily oral gavage with
the above drugs for 2 and 4 weeks at 70 days post-infection to
determine drug efficacy in chronic established infections.
To determine the length of daily exposure required to effect cidality,
3 ml of Mtb cell culture at OD[650] of 0.2 was diluted 100-fold and
exposed to test compounds as described above. Cells were exposed to
compound for 3 or 6 hours on a daily basis after which test compounds
were removed by centrifugation (4,000 g, 10 min) and washing with PBS 3
times. Washed Mtb cells were resuspended in 7H9/ADC/Tween without
compounds and incubated at 37°C for 21 hours and 18 hours,
respectively. Control cells were continually exposed to test compound
without daily washing. This process was repeated on a daily basis for 7
days after which appropriate dilutions were plated on 7H11/OADC plates.
Similarly, to determine the daily exposure required to achieve
bacteriostasis during macrophage infection, J774 cells (1× 10^5
cells/well) were seeded in 24 well plate and infected with Mtb at an
MOI 1 and exposed to test compounds as described above. Infected cells
were continually exposed to compound or for daily exposure periods of 3
or 6 hours followed by washing of monolayers 3 times with PBS and
DMEM/FBS medium replacement for a total of 7 days. Cell lysis and
plating was as described above.
To compare the level of guanine uptake of in vitro cultures of Mtb to
Mtb growing inside host macrophages, ^14C-guanine was fed to 10^8 Mtb
cells derived from early log-phase (OD[650] 0.2) 7H9/ADC/Tween culture
or from a similar number of Mtb cells derived from 5-day infected J774
cells lysed with deionized sterile water. After 3 days at 37°C, cells
were harvested by centrifugation, washed 3 times, a small aliquot used
for CFU enumeration by plating on 7H11/OADC agar plate and the
remaining cells resuspended in scintillation liquid to determine
guanine incorporation by scintillation counting.
Mtb IMPDH activity and inhibition assays
Enzyme activity and inhibition assays were performed with recombinant
truncated Mtb IMPDH where amino acids 126 to 251, corresponding to two
CBS domains on the native enzyme were substituted by two glycine
residues. Truncated Mtb IMPDH activity was monitored by the increase in
absorbance at 340nm, due to product NADH formation (ε[340nm]NADH: 6220
M^−1cm^−1) at 25°C in activity buffer (Tris HCl 50mM, KCl 150mM, TECEP
1.5mM, pH 8.0). The K[M] values of substrates IMP (70 ± 2 µM) and NAD
(937 ± 62 µM), forward reaction k[cat] (0.67 ± 0.01 s^−1) and NAD^+
substrate inhibition (10 ± 0.8 mM) of truncated Mtb IMPDH showed no
difference when compared to native IMPDH^[285]12. Inhibition assays
were performed on activity buffer in presence of Mtb IMPDH 1 µM, IMP
0.5 mM, NAD^+ 1 mM, in final 50 µL volume. Compound 1, in 100% DMSO
solution, was varied from 3 nM to 800 µM. All data points, including
controls, contained equal final volume of DMSO (2 µL). All reactions
were performed in triplicates. The fractional activity as a function of
the inhibitor concentration was fitted to the equation: v[1]/v[0] = 1/1
+ ([I]/IC[50]) for IC[50] value determination using SigmaPlot v.12.
K[i] constants were determined by some modifications to the above
protocol. Specifically, the enzymatic activity was measured by a
continuous spectrophotometric assay ^[286]10 in a 200 µl reaction
mixture that contained 50mM Tris HCl buffer, pH 8.0, 150mM KCl, 1mM
DTT, 1mM EDTA, 3mM NAD^+ and 1.25mM IMP (all chemicals were purchased
from Sigma-Aldrich). After 1 minute of pre-incubation, the reaction was
started by adding 0.5 micrograms of either the Mtb or human enzyme and
the increase in the absorbance at 340 nm, caused by the reduction of
NAD+ to NADH (ε[340] = 6220 M^−1 cm^−1), measured. The assay was
performed in quartz cuvettes with a Varian Cary 50-BiO UV-visible
spectrophotometer equipped with a temperature controlled cuvette
holder. The mechanism of inhibition and the K[i] of compound 1 against
Mtb GuaB2, was determined by analysis of the initial velocity data
plotted against the substrate concentration ([287]Fig. 3 panel d). The
data were fitted to equation describing the uncompetitive inhibition
model using Sigma Plot-Enzyme Kinetics Module 1.3. The concentrations
of compound 1 were varied from 0 to 1 µM. Data points were obtained
from two independent experiments. In the case of human IMPDH, the
concentration of compound 1 required to reduce the fractional enzyme
activity to half of its initial value (IC[50]) was calculated plotting
the enzyme fractional activity against the logarithm of inhibitor
concentration ([288]Fig. S2), and fitting the curves to a dose response
curve ([289]Equation 2):
[MATH: y=min+((max−min)/(1+10(LogIC50−x))) :MATH]
(Eq.2)
in which y is the fractional activity of the enzyme in the presence of
inhibitor at concentration [I], max is the maximum value of y observed
at [I]=0, and min is the minimum limiting value of y at high inhibitor
concentrations.
Chemistry
Compound syntheses and analytical data are described in the
[290]supplemental information.
Cloning, expression and protein purification
The M. thermoresistible GuaB2 gene was amplified from genomic DNA and
cloned into the pHat2 vector without the 2 CBS domains and a
Glycine-Glycine linker connecting the two parts of the catalytic region
(Mth IMPDH ΔCBS). The Mth IMPDH ΔCBS protein was expressed in BL21 DE3
(NEB) cells at 37°C until the OD[600] measured 0.6, then the
temperature was reduced to 18°C and IPTG was added at a final
concentration of 500 µM. Cells were left growing overnight. Cells were
harvested by centrifugation, resuspended in 50 mM Hepes pH 8.0, 500 mM
NaCl, 5% glycerol, 10 mM beta-mercaptoethanol, 20 mM imidazole. Lysis
was performed using an Emulsiflex cell disruptor (Avastin).
Clarification of the lysate was achieved by high-speed centrifugation
and filtration through a 0.45 µm filter. The clarified supernatant was
then applied to a Hi-Trap IMAC FF column (GE Healthcare) charged with
Nickel. The bound protein was eluted with lysis buffer + 250 mM
imidazole. Overnight dialysis into lysis buffer – imidazole was
performed, including incubation with TEV protease to remove the
N-terminal His-Tag. Then, in order to remove both uncleaved protein and
protease, the sample was passed through a gravity flow Nickel column.
The flow-through from this step was concentrated and injected onto a
Superdex 200 gel filtration column pre-equilibrated with 20 mM Hepes pH
8.0, 500 mM NaCl, 5% glycerol, 1 mM TCEP. Elution fractions were
collected and concentrated to 12.5 mg/mL for crystallization.
Chemoproteomics
The chemoproteomic affinity capturing experiments were performed as
previously described ^[291]28, [292]29. Briefly, sepharose beads were
derivatized with 5 at 1mM concentration, and beads were washed and
equilibrated in lysis buffer (50 mM Tris-HCl, pH 7.4, 0.4 %
Igepal-CA630, 1.5 mM MgCl[2], 5 % Glycerol, 150 mM NaCl, 25 mM NaF, 1
mM Na[3]VO[4], 1 mM DTT, and one Complete EDTA-free protease inhibitor
tablet (Roche) per 25 mL). They were incubated at 4°C for 1 h either
with 0.1 mL (0.3 mg) M. bovis BCG extract or with 1 mL (5 mg) mixed
HEK293/K-562/Placenta extract, which was pre-incubated with compound or
DMSO (vehicle control). Beads were transferred either to Filter plates
(Durapore (PVDF membrane, Merck Millipore) or to disposable columns
(MoBiTec), washed extensively with lysis buffer and eluted with SDS
sample buffer. Proteins were alkylated, separated on 4–12 % Bis-Tris
NuPAGE (Life technologies) and stained with colloidal Coomassie. Gel
lanes were cut into three slices and subjected to in-gel digest using
LysC for 2 h and trypsin overnight^[293]28. Digestion, labeling with
TMT isobaric mass tags, peptide fractionation, and mass spectrometric
analyses were performed^[294]51. Proteins were quantified by isobaric
mass tagging and LC-MS/MS. The proteins.fasta file for M. bovis BCG was
downloaded (May 11th 2011) from
[295]http://genome.tbdb.org/annotation/genome/tbdb/MultiDownloads.html
and supplemented with the sequences of bovine serum albumin, porcine
trypsin and mouse, rat, sheep and dog keratins. Decoy versions of all
proteins were created and added. The search database contained a total
of 11,492 protein sequences, 50 % forward, 50 % reverse. Protein
identification and quantification was performed^[296]52. Proteins
identified with >1 unique peptide matches were considered for further
data analysis. Apparent dissociation constants were determined by
taking into account the protein depletion by the beads ^[297]29. Raw
data tables for the chemoproteomics experiments can be found in the
[298]Supplementary Table 4 to 9.
Crystallization, compound soaking and X-ray data collection
The Mth IMPDH ΔCBS protein crystallized in 2 µl hanging drops in 1:1
ratio with 100mM sodium acetate pH 5.5, 200mM calcium chloride, 8–14%
iso-propanol. Crystals appeared after 24 hours and grew to full size
within a week. Crystals were soaked overnight in drops of well solution
+ 5mM IMP and 5mM Compound 1 dissolved in water or for 3 days in 1mM
Compound 6 solubilized in 100% DMSO. Cryoprotected crystals were passed
through drops containing well solution + 25% glycerol, and were
subsequently flash-frozen in liquid nitrogen. Data were collected from
the crystals at Diamond Light Source beamline I03 (Compound 1) and I04
(Compound 6).
Structure solution, ligand fitting and refinement
Data were processed using XDS ^[299]53 and Pointless (ccp4). To solve
the structure molecular replacement was performed with Phenix Phaser
^[300]54 using a previously solved IMP-bound Mth IMPDH ΔCBS structure
as a probe, with the NAD site empty (unpublished data). Refinement was
performed using Phenix.refine and manually in Coot ^[301]55. IMP and
compound 1 were sequentially fitted into the density using the
LigandFit function of Phenix and the structures were manually refined
further using Coot. Final R/Rfree scores obtained were 0.21/0.20 for
Compound 1 and 0.20/0.19 for Compound 6 respectively.
Information regarding the crystallographic statistics can be found in
[302]Table S10. All figures were made using Pymol (Schrodinger) and
Coot. Protein-ligand interactions were analyzed using Arpeggio (H. Jubb
– unpublished software).
Structures have been deposited in the protein data bank (PDB) as 5K4X
for IMPDH:IMP:Compound 1 and 5K4Z for IMPDH:IMP:Compound 6 complex
structures.
Mouse pharmacokinetics and bronchoalveolar lavage (BAL) studies
Test compound was dosed to female C57BL/6 mice (n=3) orally by gavage
as a fine suspension at 5, 30 and 100mg/kg free base (dose volume:
10mL/kg; Dose vehicle: 1% carboxy-methyl cellulose (CMC). Female
C57BL/6 mice were chosen as these represent the sex and strain used for
the in vivo tuberculosis efficacy models. Blood samples were taken from
the tail vein of each mouse at pre-determined time intervals post-dose,
mixed with two volumes of distilled water and stored frozen until
analysed using UPLC/MS-MS. Pharmacokinetic parameters were derived from
the blood concentration time curve using PK Solutions software v 2.0
(Summit Research Services, USA).
For the bronchoalveolar lavage studies, mice were dosed at 100mg/kg
freebase as above. The BAL fluid were extracted following tracheostomy
where a small medial incision was made, and an Insyte IV catheter (20G
– Becton Dickinson, UK) was inserted up to 1.5 cm inside the trachea
and tied with a suture to avoid leakage during BAL sampling. Both lungs
were flushed with a total 1.0 mL (0.5 mL X 2) of ice cold phosphate
buffered saline (PBS) and aspirated immediately after administration.
Aspirated volumes were recorded exactly and the samples were stored on
ice until centrifugation. Blood samples were centrifuged at 3000 rpm
for 5 min whilst BAL samples were centrifuged at 1500 rpm for 5 min as
well. Plasma and clean BAL were transferred into Eppendorf tubes, which
were stored frozen in the up-right position prior to analysis as
detailed above. The epithelial lung fluid (ELF) drug concentration was
determined as shown below: ELF drug concentration= BAL fluid drug
concentration * (plasma urea concentration/BAL fluid urea
concentration) after the method of Laohavaleeson et al ^[303]56.
Mouse, rabbit and human granuloma sample analysis
For rabbit studies, specific pathogen-free, individually housed female
NZW rabbits, weighing 2.2 to 2.6 kg, were used for aerosol infection by
M. tuberculosis HN878, as previously described ^[304]57, since it
generates a representative range of human-like lesions in infected
rabbits. Briefly, rabbits were exposed to M. tuberculosis-containing
aerosol using a nose-only delivery system. Three hours post-infection,
rabbits were euthanized, and serial dilutions of the lung homogenates
were cultured on Middlebrook 7H11 agar plates to enumerate the number
of bacterial colony forming units (CFUs) implanted in the lungs. The
infection was allowed to progress for 16 to 20 weeks, at which point
the animal were euthanized to dissect uninvolved lung pieces and
lesions as previously described^[305]58. Dissected lung tissue and
lesions were weighed, categorized as uninvolved lung, cellular
granuloma, caseous/necrotic granuloma, or cavity caseum. Each sample
was homogenized in approximately 5 volumes of phosphate buffer saline
(PBS), and stored at −80°C until analysed.
Human samples were derived from lobectomies at National Masan Hospital.
Immediately following surgery, the lung tissue was sterilely dissected
into individual lesions and uninvolved lung tissue. Larger lesions
(50–150mg, individually weighed) were separated into caseum and lesion
wall when possible. All samples for metabolite analysis were snap
frozen in liquid nitrogen.
Mouse organ samples were obtained from three uninfected C57BL/6 mice
and flash frozen on dry ice. J774 cell samples were obtained by washing
monolayers of cells with PBS, scraping of cells, harvesting, removal of
PBS supernatant and flash freezing the cell pellet on dry ice.
Supplementary Material
1
[306]NIHMS967272-supplement-1.pdf^ (1MB, pdf)
Acknowledgments