NU-6027 Inhibits Growth of Mycobacterium tuberculosis by Targeting Protein Kinase D and Protein Kinase G

Article abstract of DOI:10.1128/AAC.00996-19

Tuberculosis (TB) is a global health concern, and this situation has further worsened due to the emergence of drug-resistant strains and the failure of BCG vaccine to impart protection. There is an imperative need to develop highly sensitive, specific dia

Full text of DOI:10.1128/AAC.00996-19

AAC Accepted Manuscript Posted Online 8 July 2019
Antimicrob. Agents Chemother. doi:10.1128/AAC.00996-19
Copyright © 2019 American Society for Microbiology. All Rights Reserved.
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NU-6027 inhibits growth of Mycobacterium tuberculosis by targeting Protein Kinase D
and Protein Kinase G
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Saqib Kidwai1, Rania Bouzeyen2, Sohini Chakraborti*3, Neha Khare*4, Sumana Das*5,
Tannu Priya Gosain1, Assirbad Behura6, Chhuttan Lal Meena7, Rohan Dhiman6, Makram
Essafi2, Avinash Bajaj4, Deepak Kumar Saini5, Narayanaswamy Srinivasan3, Dinesh
Mahajan7 and Ramandeep Singh1#
*equal contribution
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Tuberculosis Research Laboratory, Vaccine and Infectious Disease Research Centre,
Translational Health Science and Technology Institute, NCR Biotech Science Cluster,
Faridabad, Haryana- 121001, India.
2 Institut Pasteur de Tunis, LTCII, LR11 IPT02, Tunis, 1002,Tunisia. Université Tunis El
Manar, Tunis, 1068, Tunisia.
3 Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012.
4 Laboratory of Nanotechnology and Chemical Biology, Regional Centre for Biotechnology,
NCR Biotech Science Cluster, PO Box # 4, Faridabad, Haryana, India.
5 Department of Molecular Reproduction, Development and Genetics, Biological Sciences
Building, Indian Institute of Science, Bangalore, India.
6 Laboratory of Mycobacterial Immunology, Department of Life Science, National Institute
of Technology, Rourkela, Odisha, India.
7 Drug Discovery Research Centre, Translational Health Science and Technology Institute,
NCR Biotech Science Cluster, Faridabad, Haryana- 121001, India.
#Corresponding author Mailing address: NCR Biotech Science Cluster, 3rd Milestone,
Faridabad-Gurugram Expressway. PO Box # 4. Faridabad – 121001.
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Running Title: Mechanism of action of NU-6027 against M. tuberculosis
Keywords: Mycobacterium tuberculosis, NU-6027, apoptosis, serine threonine protein
kinases
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ABSTRACT
Tuberculosis (TB) is a global health concern and this situation has further worsened due to
the emergence of drug resistant strains and failure of BCG vaccine to impart protection.
There is an imperative need to develop highly sensitive, specific diagnostic tools, novel
therapeutics and vaccines for the eradication of TB. In the present study, a chemical screen of
pharmacologically active compounds library was performed to identify anti-mycobacterial
compounds. The phenotypic screen identified few novel small molecule inhibitors including
NU-6027, a known CDK-2 inhibitor. We demonstrate that NU-6027 inhibits Mycobacterium
bovis BCG growth in vitro and also displayed cross-reactivity with Mycobacterium
tuberculosis protein kinase D and protein kinase G. Comparative structural and sequence
analysis along with docking simulation suggest that the unique binding site stereochemistry
of PknG and PknD is likely to accommodate NU-6027 more favorably in comparison to other
M. tuberculosis Ser/Thr protein kinases. Further, we also show that NU-6027 treatment
induces the expression of pro-apoptotic genes in macrophages. Finally, we demonstrate that
NU-6027 inhibits M. tuberculosis growth in both macrophage and mice tissues. Taken
together, these results implicate that NU-6027 may be optimized further for the development
of anti-mycobacterial agents.
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Introduction
M. tuberculosis is a major global concern with an estimated 10 million incident rates and
approximately 1.6 million deaths in 2017 according to the World Health Organisation
(WHO) Report (1). Approximately, one-third of the world population is latently infected with
M. tuberculosis. However, 5-10% of these latently infected individuals develop active disease
due to deterioration of their immune response (2). The incidence of TB associated deaths is
higher in patients with other communicable and non-communicable diseases (3). The 6-9
months duration of TB chemotherapy and poor patient compliance has led to the emergence
of drug-resistant strains. Multidrug drug resistant (MDR)-TB is defined as strains harboring
resistance to first line TB drugs, isoniazid and rifampicin (4). MDR-TB that develops
additional resistance to fluoroquinolones and one of the injectable second-line TB drug is
defined as extensively drug resistant (XDR)-TB (4). The treatment of individuals with MDR-
TB and XDR-TB requires combination of second and third line TB drugs that are expensive,
more toxic and less effective (5). This duration of medication is between 9-18 months and if
the disease progresses into totally drug resistant (TDR)-TB, TB becomes almost untreatable.
Therefore, the existing regimen for both susceptible and resistant TB needs to be simplified
and shortened.
High throughput screening is the mainstay of drug discovery and has led to the
identification of different scaffolds that are currently in different stages of clinical trials (6-9).
The current methods of screening for identification of novel scaffolds are either target-based
or phenotypic-based. However, small molecules identified through target-based screening
have achieved limited success due to lack of translation from in vitro enzymatic activity to
whole cell activity. In contrast, phenotypic screening has resulted in the discovery of various
small molecules that possess a novel mechanism of action. The novel chemical entities
identified by phenotypic screening fulfill essential criteria such as cell permeability and
provide structures that can be optimized for enhanced potency, better tolerability and
pharmacokinetic properties. Phenotypic screening along with sequencing of bacterial
genomes isolated from resistant strains has identified several compounds such as bedaquiline,
Q203, nitroimidazoles, SQ-109 and Btz-403 in TB drug discovery pipeline (10-15). Among
these, bedaquiline and delamanid have been recently approved by FDA for the treatment of
individuals with MDR-TB (16, 17). Despite their identification, there is an urgent need to
design a regimen that (i) can shorten the duration of therapy, (ii) minimize drug-drug
interaction and (iii) is active against drug-resistant and latent bacteria (5, 6, 9).
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In the present study, we have performed phenotypic screening using a library of
pharmacologically active compounds (Sigma Lopac1280) to identify inhibitors against M.
tuberculosis. Using M. bovis BCG as a host strain, we identified the lead compound, NU-
6027, 4-cyclohexyl, 2-6-diamino-5-nitrosopyrimidine, that displayed an MIC99 value of 1.56
M. We demonstrate that NU-6027 inhibits autophosphorylation activity associated with
Ser/Thr protein kinases (STPKs), protein kinase D (PknD) and protein kinase G (PknG) in a
dose-dependent manner. Computational structural studies involving molecular docking
simulation, binding site and sequence comparison analysis explain the possible molecular
mechanism of specificity of NU-6027 towards PknD and PknG. Further, we show that NU-
6027 induces apoptosis and also inhibits the growth of intracellular M. tuberculosis in both
macrophages and mice model of infection. These findings reinstate the fact that modulation
of host and bacterial signaling pathways is an attractive strategy for the design of novel anti-
tubercular agents.
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Materials and Methods
Strains, growth conditions and compound library screening: M. tuberculosis H37Rv and
M. bovis BCG were cultured in 7H9 medium supplemented with 0.05% tween-80 (v/v), 0.2%
glycerol (v/v) and 10% ADS as described previously (18). A compound library comprising
1280 pharmacologically active compounds was purchased from Sigma Aldrich. Preliminary
screening was performed against M. bovis BCG at a single concentration of 10 M. In
screening experiments, isoniazid was included as a positive control. The identified
preliminary hits were re-evaluated for anti-mycobacterial activity and MIC99 values were
determined as described previously (18). For in vitro growth inhibition experiments, early-log
phase cultures of M. bovis BCG were exposed to different concentrations of either NU-6027
or INH for 4 days or 7 days. At designated time points, 10.0-fold serial dilutions of untreated
or drug-treated cultures were prepared and plated on MB7H11 supplemented with 10%
OADS.
Chemical synthesis: The experimental details for the synthesis of parent compound, NU-
6027, and its derivatives are mentioned in the supplementary information (Supplementary
Text). The structural analysis and characterization of the synthesized compounds were
performed by 1H/13C-NMR spectroscopy and mass spectrometry. The corresponding
analytical data is provided along with experimental details in the supplementary information
(Supplementary Text).
Purification of M. tuberculosis STPK enzymes: The expression constructs for PknA, PknB,
PknD, PknE, PknF, PknG, PknH, PknJ and PknL were kind gift from Dr. Yogendra Singh,
University of Delhi. The overexpression plasmid for PknK of M. tuberculosis was a generous
gift from Dr. Vandana Malhotra, University of Delhi. Escherichia coli Arctic strain was used
for overexpression and purification of the recombinant proteins. The expression of
recombinant proteins was induced at 18oC, 180 rpm for 18-24 hrs by the addition of IPTG as
per standardized conditions. The induced cultures were harvested, resuspended in lysis buffer
and STPK’s were purified using either Ni-NTA or GST- based affinity chromatography as
per manufacturer’s recommendations. Purified GST-tagged proteins were concentrated and
dialyzed in 1x PBS containing 20% glycerol. Purified (His)6-tagged proteins were dialyzed in
buffer containing 50 mM Tris-Cl, pH 8.0, 50 mM NaCl, 0.1 mM DTT, 50% glycerol. The
purified and dialyzed recombinant proteins were stored at -20°C till further use.
In vitro kinase assays for STPKs: (His)6-PknA, (His)6-PknB, (His)6-PknD, (His)6-PknJ,
(His)6-PknL GST-PknG and GST-PknK enzymes were assayed for autophosphorylation
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activity in assay buffer (100 mM PIPES, pH 7.0, 80 mM NaCl and 20 mM MgCl2) containing
1-2μCi of γ32P-ATP for 10-45 minutes at 25°C (19, 20). Autophosphorylation assays of GST-
PknE, GST-PknF and GST-PknH were performed in buffer containing 25 mM Tris-Cl, pH
7.4, 5 mM MgCl2, 2 mM MnCl2 and 1 mM DTT for 30 minutes at 37°C (19). The auto-
phosphorylation reactions were stopped by the addition of 5x SDS sample loading buffer,
resolved on SDS-PAGE and analyzed by phosphor imaging using Typhoon 9210 imager (GE
healthcare). To determine the effect of NU-6027 on the activity of STPK’s,
autophosphorylation assays were performed either in its presence or absence. Image J
software was used to determine the extent of autophosphorylation of STPK’s in vitro.
In silico studies:
A combination of chemoinformatics and bioinformatics based analysis were performed to
understand the molecular recognition between NU-6027 and M. tuberculosis STPKs. These
studies can be broadly classified under three major steps as elaborated below:
(a) Input selection, preparation and analysis of available data: This step pertains to
selection and preparation of the appropriate protein-ligand complex structure for docking
analysis. The chemical information about NU-6027 was fetched from databases such as
PubChem, BindingDB and DrugBank (21-23). The crystal structure of human CDK2 bound
to NU-6027 solved at 1.85Å is available in the Protein Data Bank (24) with code: 1E1X (25,
26). NU-6027 is bound to the ATP binding site of human cyclin-dependent kinase 2 (CDK2)
in 1E1X, where the DFG loop of the kinase domain is in “in” conformation. This structure
has been used for comparative analysis in our study. Our initial choice for experimental
structures of STPK’s from M. tuberculosis which have been used for structural analyses were
guided by the following selection criteria which assures the reliability of the structure and
subsequent prediction: (i) The available structure of the kinase domain must be solved in holo
form (bound to an inhibitor in the ATP binding site of the kinase) with DFG loop in “in”
conformation, (ii) There should not be any missing residues in the electron density map of
ATP binding site in the protein, and/or (iii) The resolution of the solved structure should be
better than 2.5Å. At the time of our study, among the 11 M. tuberculosis STPKs,
experimental structures of inhibitor bound protein which satisfied our selection criteria were
available only for PknG (PDB code: 2PZI, 2.4Å) and PknB (PDB code: 5U94, 2.2Å). While
the kinase domain in 2PZI is reported with the DFG loop of the kinase domain in “in”
conformation (27), the information on the conformational state of the kinase domain in 5U94
(Wlodarchak, N. et al., to be published according to the Protein Data Bank entry) is not
explicitly available. The superimposition of kinase domain from different crystal structures of
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ligand bound PknB complexes (1MRU, 1O6Y and 2FUM) onto the kinase domain of PknB
in 5U94 suggested that the DFG loop in the later is in “in” conformation (28-30). This
pairwise superimposition was performed using Protein Structure Alignment module in
Maestro version 11.01.011 (Schrodinger LLC). The three crystal structures with the PDB
codes 1E1X (CDK2), 2PZI (PknG), 5U94 (PknB) were prepared using Protein Preparation
Wizard at pH 7.0 (as per the experimental assay condition) (31). The chemical structure of
NU-6027 was prepared using LigPrep (Schrödinger, LLC) before docking it into the protein
binding site. These prepared structures were used for subsequent structural analysis. In this
part of the study, OPLS3 force field was used (32).
To further enhance our understanding on NU-6027’s specificity towards PknG and
PknD, we extended our computational study to PknA (PDB code: 4OW8, an apo kinase
domain of PknA; 2.03Å and PknD) (33). The experimental structure of PknD kinase domain
is unavailable and was therefore modelled using PknD surrogate structure (i.e., structure of
PknB L145M/V155M/D33L mutant; PDB code: 3F69; 2.8Å) as template (34). The PknA
structure and PknD model was prepared in the similar way as previous structures were
prepared. The chemical structure of NU-6027 was docked into the binding pocket of PknA
and PknD model using an advanced version of OPLS3 force-field, viz., OPLS3e (35).
OPLS3e was also used for building the PknD model for docking studies.
(b) Structural analysis: This analysis of our study can be further sub-classified as under:
(i) Binding site analysis in the crystal structure of the proteins: In order to assess the local
similarity in the binding site of proteins, pairwise sequence alignment based on the structural
superimposition of binding site residues was performed (using binding site and sequence
alignment module availed through Schrӧdinger suite).
(ii) Prediction of binding pose of NU-6027 in the M. tuberculosis STPKs (PknG, PknB,
PknD, and PknA): To predict the binding pose of NU-6027 in the ATP binding site of the
mentioned kinases, we employed flexible binding site – flexible ligand docking simulation
using Induced Fit Docking (36) protocol of Schrodinger suite with extended sampling
settings (37). The best pose among all the reported output poses was selected based on
detailed analysis which involved assessment of retention of crucial interactions between the
ligand and hinge residues of the protein kinase domain. Since hydrogen bonds are known to
confer specificity to protein-ligand interactions so propensity of occurrence of a hydrogen
bond between a particular amino acid residue and the ligand among all the shortlisted poses
was calculated (38). Also, the propensity of similar poses (similar with respect to mutual
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conformation and orientation of the ligand in the respective protein binding site) predicted
throughout the entire set of output poses were taken into consideration for selecting the best
representative docked model. Comparative analysis of hydrophobic contacts was performed
on the best poses of NU-6027 in respective proteins. The criteria used to detect different non-
covalent interactions between the protein and ligand throughout the study is summarized in
Table S1. The nitrogen, oxygen, hydrogen, and sulfur atoms of the proteins and ligands are
depicted in blue, red, white, and yellow colour, respectively.
(iii) Comparative modelling: Comparative structural models of PknD - wild type (WT)
sequence was built using PknD surrogate structure (i.e., PknB L145M/V155M/D33L mutant;
3F69) as template (34). Chain B of 3F69 has fewer missing residues in the electron density
map as compared to chain A. Hence chain B of 3F69 was preferred to be used as template for
building of PknD-WT model. The protein chains in 3F69 belong to a PknB construct where
two ATP contacting residues have been mutated (L145M and V155M) to mimic PknD’s ATP
binding site. The other mutation, D33L, is a stabilizing mutation at a site far away from the
ATP binding pocket. Prime homology modelling tool was used for model building and only
the non-template loop in binding site was refined (39, 40). The quality of the model was
assessed using PROCHECK and observed to be satisfactory (data not shown) (41).
(c) Sequence analysis: In order to understand the possible reasons for NU-6027 specificity
towards PknG and PknD enzyme, we also performed multiple sequence alignment (MSA) of
amino-terminus stretch and kinase domain of all the 11 M. tuberculosis STPKs and human
CDK2 using MUSCLE (42). The MSA was viewed using ESPript3.0 (43). We also
performed Structure based multiple sequence alignments (SBMSA) of kinase domains of M.
tuberculosis STPKs employing PROMALS3D tool. For SBMSA analysis, experimental
structures of the proteins were used wherever available and for remaining STPK’s sequences
were used (44).
Macrophage experiments: The THP-1 human monocyte cell line was maintained in RPMI
1640 medium containing 10% fetal bovine serum (FBS) as per standard protocols (45). THP-
1 monocytes were differentiated by the addition of 20 ng/ml phorbol 12-myristate 13-acetate
(PMA) for 48 hrs. Post-differentiation, THP-1 cells were washed twice with 1x-phosphate
buffered saline (PBS) and rested for 1 day in RPMI medium. Cell viability assays were
performed using cell proliferation reagent, WST-1 as per manufacturer recommendations.
For intracellular killing experiments, differentiated THP-1 macrophages were infected with
either M. bovis BCG or M. tuberculosis as previously described (18). Briefly, 24 hrs post-
infection, macrophages were overlaid with RPMI medium containing NU-6027 or INH for 4
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days. At designated time points, macrophages were lysed and 10.0 fold serial dilutions were
plated on MB7H11 plates at 37oC for 3-4 weeks. For qPCR analysis, total RNA was isolated
from untreated and NU-6027 treated macrophages using Qiagen RNAeasy kit. DNase-I
treated RNA was subjected to cDNA synthesis using Superscript III reverse transcriptase.
The relative expression levels of gene of interest were calculated after normalization to the
expression levels of housekeeping gene, GAPDH as per standard protocol. The list of primers
used for qPCR analysis is shown in Table S2.
Analysis of autophagy by fluorescence microscopy: LC3 puncta formation in untreated and
NU-6027 treated macrophages was visualized by confocal microscope as previously
described (18). In these experiments, macrophages were incubated with NU-6027 for 6 hrs or
24 hrs. In few experiments, 50 nM Bafilomycin A (Baf-A1) was added to macrophages for 3
hrs before the completion of experiment. At designated time points, macrophages were fixed,
stained and images were captured using confocal scanning laser microscope (Leica
Microsystems).
Analysis of apoptosis by flow cytometry: For detection of apoptosis, mock or infected
macrophages with or without NU-6027 treatment were harvested. The macrophages were
washed with 1x binding buffer (100 mM HEPES-pH 7.4, 1.4 M NaCl, and 25 mM CaCl2
solution) and stained with a mixture of PE-Annexin V and 7-AAD at room temperature. After
15 minutes of incubation, 400 µl of 1x binding buffer was added and data was acquired using
FACS Caliber (Becton Dickinson). The acquired data was subsequently analysed using
FLOWJO 7.6.1 software.
Ethics Statement. All animal studies were performed in accordance with committee for the
purpose of control and supervision on animals (CPCSEA) guideline and approved from the
Institutional animal ethics committee of THSTI and ICGEB. All experiments involving M.
tuberculosis were performed in BSL-3 laboratory at Tuberculosis aerosol challenge facility,
ICGEB.
Pharmacokinetics and dose tolerance study. NU-6027 was resuspended in 1%
carboxymethyl cellulose to prepare a dosing formulation of 10 mg/ml strength. For drug-
tolerability experiments, 100 mg/kg/day of NU-6027 was orally administered for 5
consecutive days. Various parameters such as body weight, fur loss, body temperature and
mice activity were monitored for 7 days post-drug administration. For pharmacokinetic
studies, 4-6 weeks old BALB/c mice were orally administered NU-6027 at a dose of 100
mg/kg. At designated time points (3 mice per group) plasma was prepared and subjected to
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LC/MS analysis. The detail of pharmacokinetics experiment is provided in the supplementary
information (Supplementary Text).
Mouse Infection experiments. 4-6-week-old BALB/c mice were infected with
approximately 100 CFU of M. tuberculosis via aerosol route. For efficacy studies, 28 days
post-infection, rifampicin or NU-6027 was administered orally at a dose of 10 mg/kg or 100
mg/kg, respectively for 5 days a week. At designated time points lungs and spleens were
homogenized in normal saline and 10.0 fold serial dilutions were plated on MB7H11 plates at
37oC for 3 – 4 weeks.
Statistical analysis: All statistical analysis was performed using Graph Pad Prism software.
The differences between indicated groups were considered significant with p < 0.05.
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Results
Phenotypic screening results in identification of NU-6027 as the lead compound: M.
bovis BCG is widely used as a host strain for the screening of small molecule libraries as it
can be cultured within a BSL-2 environment and it shares about 99% similarity with M.
tuberculosis genome (46). Previous studies have demonstrated that hit rates obtained using
M. bovis BCG are similar to those obtained in M. tuberculosis (7, 18, 47). In the present
study, we screened a library of pharmacologically active compounds (Sigma Lopac1280
)
comprising 1280 compounds to identify small molecule antimycobacterial agents. The
preliminary screening was performed at a single concentration of 10 M in duplicates and all
plates included DMSO and INH as negative and positive controls, respectively. As expected,
no growth inhibition of M. bovis BCG was observed in the presence of DMSO while INH
yielded complete growth inhibition. We identified 20 primary hits that were able to inhibit
the growth of M. bovis BCG (Table 1). A number of these compounds have been previously
reported to be active against M. smegmatis, M. bovis BCG and M. tuberculosis (48). The
structures with already reported activity and those that possessed reactive functional groups
were eliminated for future mechanistic studies. Based on our review of available literature,
mechanism of action for anti-mycobacterial activity has not been reported in the case of 2-[3-
dihydro-(1,3,3-trimethyl-2H-indol-2-ylidene)-1-1propenyl]-3-ethyl-benzothiazolium iodide
(AC-93253 iodide), calcimycin (C7522), diphenylene iodonium chloride (D2926) and 4-
cyclohexyl, 2-6-diamino-5-nitrosopyrimidine (NU-6027). We next determined IC50 values of
these small molecules against THP-1 cell line. We did not observe any cytotoxicity in the
case of NU-6027 and AC-93253 on THP-1 macrophages at 25 M and 10 M, respectively.
However, both C7522 and D2926 showed cytotoxicity on THP-1 macrophages at a
concentration higher than 2.5 M and 0.5 M, respectively. Recently, we have reported that
pre-treatment of macrophages with calcimycin induces IL-12 production and also enhances
autophagy in P2XR7 dependent manner (45, 49). In concordance with our findings, it has
been reported that diphenyleneiodonium chloride displays bactericidal activity against
Staphylococcus aureus and M. tuberculosis (50).
In the following sections, we discuss activity results of NU-6027 against
mycobacteria in vitro, in macrophages and mice tissues. The chemical structure of NU-6027
consists of a 2,6 diamino pyrimidine linked to cyclohexyl ring by an alkoxy group. We
hypothesized that the nitroso group attached to the pyrimidine core of NU-6027 is
pharmacologically relevant and critical for the observed in vitro activity. To test this
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hypothesis, we synthesized the parent compound (a) along with few other truncated
analogues and evaluated them for anti-mycobacterial activity in vitro (Fig. 1a). As expected,
the parent compound (a) displayed MIC99 value of 1.56 M against M. bovis BCG. In
concordance with our hypothesis, we observed that the removal of NO group (b) completely
abolished the in vitro activity associated with NU-6027 (Fig. 1a). The other synthesized
truncated analogues (c and d) were also inactive in our in vitro activity assays (Fig. 1a). In
conclusion, this limited analog screening did highlight the importance of nitroso group for the
anti-mycobacterial activity of NU-6027. Next, we determined the mode of M. bovis BCG
growth inhibition by NU-6027. We observed that exposure to 10x MIC99 concentration of
NU-6027 for 7 days reduced the viable counts by approximately 4.5 folds (Fig. 1e, *p<0.05).
As expected, 70.0-fold reduction was seen in 10x MIC99 concentration of INH-treated
cultures after 7 days of exposure (Fig. 1e, **p<0.01). We also demonstrate that NU-6027 is
able to inhibit the in vitro growth of M. bovis BCG in both dose and time dependent manner
(Fig. 1f, *p<0.05 and **p<0.01).
NU-6027 inhibits PknD and PknG enzymes from M. tuberculosis: The substituted
pyrimidine ring of NU-6027 mimics the adenine ring of ATP and is involved in interactions
with critical residues in the crystal structure of CDK2 (25). Since bacterial and mammalian
kinases possess conserved architecture of ATP binding site in their kinase domain, we
hypothesized that NU-6027 might be able to target multiple cellular events regulated by M.
tuberculosis STPK’s. Numerous studies have shown that STPK’s undergoes
autophosphorylation to initiate phospho-mediated signaling (51). To test this hypothesis, we
next evaluated the ability of NU-6027 to inhibit autophosphorylation activity associated with
M. tuberculosis STPK’s. For in vitro autophosphorylation assays, M. tuberculosis STPK’s
were purified as either (His)6-tagged proteins (in the case of PknA, PknB, PknD, PknJ and
PknL) or as GST-tagged proteins (in the case of PknE, PknF, PknG, PknH and PknK),
concentrated, dialysed and stored in -80oC till further use (Fig. S1). Next, we determined the
effect of NU-6027 on the autophosphorylation activity of all purified STPK enzymes except
PknI (Fig. 2). We observed that NU-6027 inhibited autophosphorylation activity associated
with both PknD and PknG in a dose-dependent manner (Fig. 2a and 2b). As shown in Fig. 2a,
we demonstrated that NU-6027 did not affect the auto-phosphorylation activity associated
with other tested STPK’s (PknA, PknB, PknE, PknF, PknH, PknJ, PknK and PknL). These
results implicate that NU-6027 specifically inhibits PknD and PknG auto-phosphorylation
without affecting other STPK’s.
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Comparative structural analyses to understand the specificity of NU-6027 towards
PknG and PknD: As discussed in the Materials and methods section, initially, the crystal
structures of PknB (5U94) and PknG (2PZI) were selected for detailed computational
analysis. The structure of PknB in 5U94 served as a representative among the 8 M.
tuberculosis STPKs (PknA, PknB, PknE, PknF, PknH, PknJ, PknK, and PknL) which did not
show any inhibitory response in the presence of NU-6027. The available structure of PknG
and PknB provided an opportunity to investigate the possible reasons for NU-6027 specificity
towards PknG over PknB. The structure of human CDK2 bound to NU-6027 (PDB code:
1E1X) helped in understanding the critical interactions which are essential to elucidate kinase
activity inhibition by NU-6027. The molecular docking studies were later extended to PknD
and PknA.
(i) Binding site analysis of PknG and PknB: Pairwise structure-based binding site
sequence comparison between PknB (5U94) and PknG (2PZI) revealed that while the root
mean square deviation (RMSD) of Cα atoms of the aligned residues is 1.5Å, the sequence
identity between the binding site residues is 48% (Fig. S2a, b). These observations suggest
that the binding sites of these proteins differ in both composition and conformation of the
constituting residues. Such dissimilarities of binding site structures might confer a varying
response by different STPKs in recognition of NU-6027. To understand the probable binding
mode of NU-6027 to PknG and PknB, we next performed molecular docking analysis.
(ii) PknG-NU-6027 induced fit docking analysis: The output poses from the induced fit
docking (36) experiments were manually scrutinized and selected for further analysis as
described in Materials and methods section. We observed that in 50% and 38% of the
selected poses from PknG:NU-6027 experiment, the 5-nitroso and 6-amino substituent of
NU-6027 are engaged in hydrogen bond formation with the main chain of the hinge residues:
Val235 and Glu233, respectively. Overall, 31% of the poses are engaged in hydrogen
formation with both Val235 and Glu233. The predicted pose of NU-6027 in PknG from our
docking experiments is similar to the bound pose of AX20017, a known PknG specific
inhibitor (2PZI), with respect to placement of the hydrophobic and adenine-mimetic rings of
the two compounds (Fig. S2c, d). Interestingly, AX20017 as well as ATP analogues (PDB
code: 4Y0X, 4Y12) are also involved in hydrogen bond interactions with these amino acid
residues (52). Their equivalent residues, Glu81 and Leu83 in the case of CDK2 and Glu93
and Val95 in the case of PknB (PDB code: 1O6Y, 1MRU, 5U94) are also known to
participate in hydrogen bond formation with their respective inhibitor or the adenine ring of
ATP or ATP analogues (28, 30). Previously, it has been shown that pyrimidine and purine
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analogues which failed to engage Glu81 of CDK1/2 in hydrogen bond formation are unable
to inhibit these enzymes (25). However, pyrimidine and purine analogues that were unable to
form hydrogen bond with Leu83 displayed reduced inhibition of activity associated with
CDK1/2 enzymes (25). In addition to these interactions, we also observed that the amino
group at 6th position of the pyrimidine ring of NU-6027 forms hydrogen bond with the main
chain of Ile292 in 92% of PknG:NU6027 shortlisted poses. The Ile292 residue is unique to
the binding site of PknG and is substituted with methionine and alanine in PknB and CDK2,
respectively (Fig. S3). Interestingly, in the top ranked pose, NU-6027 is involved in hydrogen
bonding with Glu233, Val235 and Ile292 of PknG. In addition, the 2-amino substituent in the
pyrimidine ring of NU-6027 is also involved in hydrogen bond formation with the side chain
of Asp293 (Fig. 3a). This Asp293 is part of the conserved DF(/L)G motif near the activation
loop of kinases. In addition to the conserved residues, NU-6027 also forms hydrophobic
interactions with six non-conserved residues which include Ile165, Val179, Ile292 and
Gly236 from the kinase domain and Ile87 and Ala92 in the amino-terminus region (Fig. 3a).
These residues have also been demonstrated to be important for interactions between
AX20017 and PknG binding site (27).
(iii) PknB-NU-6027 induced fit docking analysis: Analysis of the induced fit docking poses
of NU-6027 in PknB showed that the functional residues such as Glu93 and Val95 are not
involved in hydrogen bond formation with NU-6027. Previously, it has been shown that
inhibitor such as G93 forms hydrogen bond with residues such as Glu59, Lys40 and Asp156
(Wlodarchak, N. et al., to be published according to the Protein Data Bank entry). However,
these residues are not involved in hydrogen bond formation in the predicted PknB:NU-6027
induced fit docked poses (Fig. 3b). The best representative induced fit docked pose of NU-
6027 in PknB binding site is predicted to be involved in hydrogen bonding with Leu17 and
Ala142 (which are not known to be crucial for enzyme inhibition).
(iv) PknD-NU-6027 induced fit docking analysis: Analysis of the induced fit docking poses
of NU-6027 in PknD-WT model showed that the two hinge residues, Arg93 and Ile95 are
involved in hydrogen bond formation with NU-6027 in 9% and 91% of the poses,
respectively. In 9% of the overall poses, both Arg93 and Ile95 are involved in hydrogen bond
formation with NU-6027. Additionally, NU-6027 is also involved in hydrogen bond
formation with main chain of Gly97 in 82% of the poses. The best representative pose of NU-
6027 in PknD binding site is predicted to be involved in hydrogen bonding with Ile95 and
Gly97 (Fig. 3c).
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(v) PknA-NU-6027 induced fit docking analysis: Analysis of the induced fit docking poses
of NU-6027 in PknA showed that the two hinge residues, Glu96 and Val98, are involved in
hydrogen bond formation with NU-6027 in some of the poses. However, both these residues
are involved in hydrogen bond formation with NU-6027 in only 0.06% of the overall poses.
The best representative pose of NU-6027 in PknA binding site is predicted to be involved in
hydrogen bond formation with Glu96 and Asp159 (Fig. 3d). Contrary to other docking
simulations, we also noticed that a part of the hydrophobic component (few carbon atoms of
the cyclohexyl ring) of NU-6027 in PknA binding site is solvent exposed in all the predicted
poses. This might be the reason for inability of NU-6027 to bind and inhibit PknA
autophosphorylation activity.
(vi) Comparative analysis of binding mode of NU-6027 in PknG with other studied
kinases: The docked poses of NU-6027 from PknG:NU-6027 and PknB:NU-6027 predicted
complexes were overlaid on to the bound pose of NU-6027 obtained from the crystal
structure of human CDK2 (Fig. 4a). We noticed that the pyrimidine ring of NU-6027 in
CDK2 and PknG occupies almost equivalent regions in their respective protein binding
pocket. Although the ligand atoms involved in pyrimidine ring mediated interactions with
PknG and CDK2 differ but the crucial interactions with the conserved ATP binding site are
retained (Fig. 4a). We observed a displacement of almost 80° between the cyclohexyl ring of
NU-6027 in PknG as compared to that in CDK2. Consequently, the cyclopropyl ring
mediated hydrophobic contacts between NU-6027 and PknG or CDK2 are distinct. As shown
in Fig. 4a, the docked pose of NU-6027 in PknB is such that the pyrimidine and cyclohexyl
rings are almost flipped by 180° with respect to the bound pose of the ligand in CDK2 which
would prohibit the establishment of relevant interactions between PknB and NU-6027.
Interestingly, in PknD:NU-6027, the pyrimidine ring of NU-6027 is placed near to the hinge
region similar to that observed in PknG:NU-6027 predicted complex (Fig. 4b). These
observations suggests that crucial interactions required for enzyme inhibition are most likely
to be retained in the case of PknD. Surprisingly, the best representative pose of NU-6027 in
PknA binding pocket also resembles the pose of NU-6027 in PknG (Fig. 4b). However, in
only 0.06% of the total PknA poses (in comparison to 31% in the case of PknG), both
functional residues in the hinge region are involved in hydrogen bond formation with NU-
6027. Moreover, we observed that the hydrophobic cyclohexane ring of NU-6027 is solvent
exposed in all the PknA:NU-6027 poses (Fig. 4c). On the contrary, the corresponding atoms
of the cyclohexane ring of NU-6027 in PknG is shielded by a set of unique residues from the
kinase domain (Gly236) and the N-terminal segment (Ile87 and Ala92) (Fig. 4d). Taken
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together, these observations are in concordance with our experimental results showing that
NU-6027 possesses dual specificity against PknD and PknG and is unable to inhibit the
autophosphorylation activity of PknA and PknB enzyme in vitro.
Sequence analysis of PknG with other STPKs: Our docking analysis revealed that the
predicted binding site of NU-6027 in PknG comprises of unique amino acid residues (Ile165,
Val179, Gly236, Ile292, Ile87 and Ala92) that are absent in PknB binding site. In
concordance, similar observations are also noted in the comparative sequence analysis of
PknG with other M. tuberculosis STPKs (Fig. S3). Sequence analysis of 491 human protein
kinases published earlier revealed that the frequencies of occurrence of Ile165, Val179,
Gly236 and Ile292 amino acid residues surrounding the PknG binding site are 0.037, 0.065,
0.088 and 0.077, respectively. Further, the combination of these four residues is not observed
in the sequence of any other human kinases (27, 53). Additionally, Ile87 and Ala92 residues
lying in the amino-terminal peptide stretch are also specific to the ligand-binding pocket of
PknG. Previously, it has been demonstrated that Ala92 and Ile87 are essential in the binding
of PknG specific inhibitors (27). Further, SBMSA analysis revealed that arrangement of two
residues, Ser99 and Val155, in PknD binding site might be important for NU-6027 specificity
(Fig. S4). The residues corresponding to Val155 of PknD are not branched chain amino acids
in other STPKs except for PknG, PknE and PknH. The corresponding residues are Ile292 in
PknG, Val156 in both PknE and PknH sequences (Fig. S4). Also, our detailed SBMSA
analysis revealed that only PknG and PknK contain a serine residue (Ser293 in PknG and
Ser110 in PknK) equivalent to Ser99 in PknD (Fig. S4).
NU-6027 enhances apoptosis in M. bovis BCG infected macrophages: M. tuberculosis is a
highly successful intracellular pathogen by virtue of its ability to inhibit several antimicrobial
mechanisms of the host. These include pathways such as generation of reactive oxygen and
nitrogen species, phagosome-lysosome fusion, autophagy and apoptosis (54, 55). Several
studies have shown that augmentation of host defensive pathways such as autophagy results
in inhibition of the growth of intracellular mycobacteria (56). Thus, we next determined the
ability of NU-6027 to induce autophagy in THP-1 cells. We did not observe any significant
increase in LC3-puncta formation in NU-6027 treated THP-1 cells at both 6 hrs and 24 hrs
post-treatment (Fig. S5a). As expected, a significant increase in LC3-puncta formation was
observed in calcimycin treated macrophages (Fig. S5b, **p<0.01). We also measured
autophagy flux in NU-6027 treated THP-1 cells by inhibiting phagosome-lysosome fusion in
the presence of Baf-A1. However, we did not observe any differences in LC3 puncta
formation in NU6027 treated THP-1 cells in the absence or presence of Baf-A1 (Fig. S5c and
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S5d). These observations demonstrate that NU-6027 is unable to induce autophagy in THP-1
macrophages.
In addition to autophagy, apoptosis is another major mechanism elicited by the
macrophages to limit multiplication of intracellular pathogens like M. tuberculosis (57).
Previously, it has been shown that NU-6027 inhibits CDK1/2, ATR enzymes and induces
apoptosis in ovarian cancer cell lines (25, 58, 59). Hence, we next sought to investigate the
ability of NU-6027 to enhance apoptosis of M. bovis BCG-infected macrophages, 24 hrs
post-treatment (Fig. 5a). We found that the percentage of Annexin-V positive cells was
significantly enhanced in M. bovis BCG-infected macrophages after treatment with NU-6027.
The percentage of Annexin-V positive cells in NU-6027 treated and BCG infected
macrophages was 35.6% and 33.6%, respectively (Fig. 5a). As shown exposure to NU-6027
enhanced percentage of apoptosis in M. bovis BCG infected macrophages to 55.6% (Fig. 5a).
Previous studies have reported that enhanced apoptosis of mycobacteria infected
macrophages is associated with upregulation of pro-apoptotic genes (60). In an attempt, to
better understand the mechanism of induction of apoptosis by NU-6027, we quantified the
transcript levels of apoptosis related genes such as NOXA and TNF-α in NU-6027 treated
macrophages (Fig. 5b). In comparison to non-treated macrophages, NOXA transcript levels
were increased by approximately 6.0-fold in NU-6027 treated macrophages (Fig. 5b,
*p<0.05). It has been reported that NOXA activation results in upregulation of signaling
pathways, which eventually results in mitochondria activation (61). Hence, we sought to
evaluate the expression of mitochondria derived pro-apoptotic proteins, apoptosis induced
factor (AIF) and endonuclease G (EndoG) in NU-6027 treated THP-1 macrophages. As
shown in Fig. 5b, we observed that the transcript levels level of AIF and EndoG were
increased by approximately 3.0 and 2.0 folds, respectively in NU-6027 treated THP-1 cells.
Interestingly, we also observed that NU-6027 increased the transcription of TNF-α by
approximately 7.0 folds suggesting the activation of apoptosis pathway (Fig. 5b, *p<0.05).
These observations indicate that NU-6027 is a potent inducer of apoptosis in macrophages by
activating these pathways.
NU-6027 is able to inhibit the growth of M. bovis BCG and M. tuberculosis in THP-1
macrophages: Previously, it has been reported that PknG is secretory in nature and promotes
survival of M. tuberculosis in macrophages by inhibiting phagosome-lysosome fusion (62-
64). Previous studies have shown that compounds modulating host apoptosis and targeting
PknG are promising anti-mycobacterial agents (27, 64-71). Since NU-6027 possessed both
these activities, we sought to assess its anti-mycobacterial activity in THP-1 macrophages
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infected with either M. bovis BCG or M. tuberculosis. Based on CFU enumeration, we
observed that NU-6027 inhibited growth of both intracellular M. bovis BCG and M.
tuberculosis (Fig. 5c and 5d). In THP-1 macrophages, pre-treatment with INH reduced the
bacterial loads by approximately 93% and 86% in the case of M. bovis BCG and M.
tuberculosis, respectively (Fig. 5c and 5d, **p<0.01). In comparison, treatment with NU-
6027 inhibited the growth of M. bovis BCG and M. tuberculosis by 78.1% and 80.1%,
respectively (Fig. 5c and 5d, *p<0.05).
We next determined whether Z-VAD-FMK, an inhibitor of apoptosis abrogates
intracellular killing activity of M. bovis BCG by NU-6027 (72). As shown in Fig. 6a, we
observed that pretreatment with Z-VAD-FMK reduced the percentage of apoptosis induced in
M. bovis BCG infected macrophages upon NU-6027 treatment by 50%. In order to further
dissect the role of induction of macrophage apoptosis and intracellular killing, we also
demonstrate that preincubation of macrophages with Z-VAD-FMK reduced the killing
activity of NU-6027 against intracellular mycobacteria (Fig. 6b, *p<0.05). These results
reaffirm previous observations that augmentation of apoptosis in infected macrophages
results in reduced intracellular mycobacterial survival.
NU-6027 is well tolerable in BALB/c mice: Next, we performed an oral single dose
pharmacokinetic study of NU-6027 in BALB/c mice to determine plasma exposure of lead
compound NU-6027. For pharmacokinetic studies, 4-6 weeks old BALB/c mice were orally
administered NU-6027 at a dose of 100 mg/kg using 1% CMC as dosing vehicle. Animals
were bled under mild anesthesia at designated time points and LC/MS analysis was
performed to determine different parameters such as AUC, Cmax, Tmax and T1/2 as described in
Materials and methods. We observed that NU-6027 was orally bio-available with AUC of 1.5
M and T1/2 of 0.96 hours (Fig. 7a and 7b). Next, we performed dose tolerance study to
determine an appropriate dose for efficacy experiments in infected animals. For drug
tolerance experiment, healthy BALB/c mice were dosed with 100 mg/kg of NU-6027 for 5
consecutive days and monitored for various health parameters. We observed that daily
administration of NU-6027 did not alter either body weight or food intake when compared to
the control group (data not shown). The animals from NU-6027 treated group and control
group were sacrificed at the end of this study and vital organs (liver, heart and kidney) were
visually inspected. The tissues from NU-6027 treated mice did not show any discoloration or
change in morphology of the vital organs when compared to controls (data not shown). These
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results indicate that a dosage of 100 mg/kg for NU-6027 in BALB/c mice is well tolerable
and suitable for an efficacy experiment to establish the required proof of concept studies.
NU-6027 inhibits the growth of M. tuberculosis in mice tissues: For efficacy experiments,
female BALB/c mice were infected with single cell suspension of log-phase cultures of M.
tuberculosis via aerosol route. After 4 weeks of infection, both NU-6027 and Rif were orally
administered at a dose of 100 mg/kg and 10 mg/kg, respectively for either 2 weeks or 4
weeks. As shown in Fig. 7c, in comparison to untreated control group, reduction in lung
bacillary load was observed in mice treated with either Rif or NU-6027. We observed that at
4 weeks post-treatment, lung bacillary loads in control, Rif-treated and NU-6027 treated mice
were log10 5.42, log10 3.90 and log10 4.52, respectively (Fig. 7d, *p<0.05 and ***p<0.001). A
similar reduction in splenic bacterial loads was also noticed in these mice after 4 weeks of
treatment. We observed that 4 weeks post-treatment, the splenic bacillary load in Rif and
NU-6027 treated mice was reduced by approximately 90.0-100.0 fold in comparison to the
bacterial loads in spleens of untreated mice (Fig. 7d, ***p<0.001). At 2 weeks post-
treatment, Rif alone reduced lung bacillary loads by 40.0 fold in comparison to untreated
animal, while oral administration of NU-6027 reduced the bacterial loads in lungs by
approximately 8.0 fold (Fig. 7c, *p<0.05 and ***p<0.001). In concordance, 2 weeks
treatment of mice with Rif or NU-6027 reduced the splenic bacillary loads by 10.0 fold and
5.0 fold, respectively, in comparison to the untreated mice (Fig. 7d, *p<0.05). Taken
together, these studies suggest that the dual mechanism of augmenting host apoptosis and
inhibiting PknD and PknG STPK’s from M. tuberculosis is a promising strategy to kill M.
tuberculosis in macrophages and mice tissues.
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Discussion.
Phenotypic screening has resulted in identification of many scaffolds that are in
clinical pipeline (6, 8). Despite the availability of increasing number of preclinical
candidates, there is still a need to develop more drugs with a novel mechanism to tackle this
global epidemic. In the present study we have performed phenotypic assays to screen a
library of pharmacology active compounds (Sigma Lopac1280) for identification of anti-
mycobacterial compounds. We identified 20 compounds that possessed anti-mycobacterial
activity and selected NU-6027 for further evaluation in this study. The other identified
molecules were given low priority, either due to their previously reported activities or
because of cellular cytotoxicity or structural features. Here, we report that NU-6027 inhibited
M. bovis BCG growth in a dose dependent manner and the nitroso functional group is
essential for the in vitro activity. These observations are in concordance with previous studies
where the Nitro functional group has been shown to be important for anti-mycobacterial
activity associated with various scaffolds (10, 11, 18).
NU-6027 is a potent inhibitor of various kinases such as CDK1/2, ATR and DNA-PK
with Ki values of 1.3/2.5 M, 0.4 M and 2.2 M, respectively (25, 73). Since protein
kinases share similar structural fold in their active sites, we hypothesized that NU-6027 might
also be able to inhibit STPK’s from M. tuberculosis. In support of our hypothesis, we
observed that NU-6027 inhibits autophosphorylation activity associated with PknD and PknG
in
a dose-dependent manner. Intriguingly, the lead compound failed to inhibit
autophosphorylation activity associated with other STPK’s including PknA and PknB.
Previously, structures with amino-pyrimidine scaffolds have been reported as inhibitors of
PknB in addition to human kinase (74, 75). Next, to understand the specificity of NU-6027,
detailed computational structural as well as sequence analyses were performed. We noticed
that in the modeled PknG:NU-6027 complex, NU-6027 is interacting with functional
residues, which are known to be involved in ATP binding. However, in the case of PknB,
interactions with these functional residues are not observed in any of the predicted poses of
PknB:NU-6027. An in-depth comparative analysis of the docked models revealed that apart
from conserved kinase domain residues, a set of six unique residues (Ile165, Val179, Gly236,
Ile292, Ile87 and Ala92) surrounding the PknG binding site are also be involved in non-
covalent interactions with NU-6027. These residues are absent in PknB binding site.
Comparative sequence analysis of 11 STPKs from M. tuberculosis confirmed that these
residues are present only in PknG and are absent in the sequences of other M. tuberculosis
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STPKs. The combination of these residues is also absent in the sequences of human kinases
as mentioned earlier. We also observed that 2-amino substituent in the pyrimidine ring of
NU-6027 is involved in hydrogen bond formation with the side chain of Asp293 in PknG.
Moreover, the binding mode of NU-6027 in PknG is such that its cyclohexyl ring can interact
with a non-conserved hydrophobic pocket which is distinct from CDK2:NU-6027 crystal
structure. These unique interactions would provide the guiding steps for further optimization
of potency and specificity of NU-6027 towards PknG. However, these interactions are solely
based on in silico studies and demand further experimental validations. Such validations
could aid in estimating the risk-to-benefit ratio for taking NU-6027 forward as an effective,
safe, and novel anti-tubercular lead molecule.
Several studies have shown that modulation of host and bacteria signaling pathways
is an attractive target to combat TB (76). To establish a successful infection, M. tuberculosis
relies on signal transduction pathways such as TCS, STPKs to sense extracellular signal and
adapt in the host through a cascade of protein phosphorylation and dephosphorylation events
(76, 77). Among STPKs PknA, PknB and PknG are required for intracellular growth and
survival (76). Both PknA and PknB are essential for M. tuberculosis growth in vitro (78, 79).
PknG has also been reported to be involved in M. tuberculosis survival in macrophages
because of its role in glutamate metabolism or via modulation of host signaling pathways
such as phagosome-lysosome fusion (62, 80, 81). In addition to these functions, PknG is also
required for M. tuberculosis growth during stationary phase, biofilm formation and establish
infection in guinea pigs and mice (64). Therefore, targeting PknG by NU-6027 would result
in inhibition of bacterial metabolism and phagosome-lysosome fusion to promote
intracellular killing.
In another study it has been shown that PknD is an essential regulator of osmo-
sensory signaling pathway and peptidoglycan architecture (82). In addition, PknD is also
required for M. tuberculosis growth in phosphate poor growth conditions and pathogenesis
(83). The results of PknD:NU-6027 docking coupled with sequence analysis revealed that
Val155 preceding the aspartic acid residue in DF(/L)G motif might be crucial for binding of
NU-6027 to PknD. Interestingly, the corresponding amino acid residue is Ile292 and Met155
in the case of PknG and PknB, respectively. We hypothesize that the straight and bulky side
chain of methionine might hinder the binding of NU-6027 in PknB binding site whereas
branched and comparatively shorter chain residues would provide optimum contacts
facilitating binding of NU-6027 in the case of PknD and PknG. In concordance, it has been
demonstrated that Met155 hinders the binding of the inhibitor KT5720 in PknB, thus
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contributing towards the specificity of KT5720 against PknD in comparison to PknB (34).
Another interesting similarity between PknG and PknD is the presence of a serine residue at
equivalent positions, 239 and 99, respectively. All other M. tuberculosis STPKs (except
PknK) has a bulkier amino acid residue at this position. Therefore, we speculate that the
presence of a serine residue in combination with branched chain residue at position 155 of
PknD (292 of PknG) might provide the specificity of NU-6027 towards PknG and PknD.
Interestingly, on the hand, our analysis hints that favorable accommodation of NU-6027 in
PknA and PknB binding pocket is unlikely which corroborates with our experimental
findings as explained earlier.
Apoptosis of infected macrophages is considered as an important innate defense
mechanism that enables the direct killing of intracellular mycobacteria by destroying its
habitat and stimulating an efficient adaptive immune response (65-67). Several studies have
shown that virulent strains of M. tuberculosis disrupt host apoptotic pathways in order to
enhance their intracellular survival (84). In our study, NU-6027 significantly enhanced the
apoptosis of M. bovis BCG infected THP-1 macrophages. We also show that NU-6027
treatment of macrophages results in induction of apoptosis related genes such as NOXA and
TNF-. We also observed that NU-6027 mediated inhibition of PknD, PknG enzymes and
apoptosis induction reduced survival of M. tuberculosis in mice tissues. These observations
are in concordance with the current notion that apoptosis of the infected phagocytes prevents
mycobacteria to establish a suitable niche for its long-term survival in the host (65). In
agreement, other studies have also identified PknG specific inhibitors such as AX20017,
amino-pyrimidine derivatives, AZD7762, R406, R406f, CYC116 and Sclerotiorin as
promising antimycobacterial agents (27, 68-71, 76). Taken together, we show for the first
time that small molecule targeting PknD and PknG has no side effects and inhibits bacterial
growth in lungs and spleens of infected mice.
Numerous reports have demonstrated that supplementation of anti-TB therapy with a
modulator of the host responses is an effective strategy against drug-resistant strain and also
reduces the risk of relapse (6). Our experimental finding that NU-6027 is able to inhibit the
growth of intracellular M. tuberculosis in mice model suggests that it can be used as an
adjunct to TB therapy. Since active TB patients also show a defect in monocytes and
macrophages apoptosis, NU-6027 can also be proposed as an apoptosis inducer during TB
chemotherapy (85, 86). Developing novel molecules, that are able to unleash the
mycobacteria-mediated blocking of macrophage apoptosis, might prove useful as a host-
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directed strategy against TB. The observed intracellular activity underscores the potential for
NU-6027 and similar STPK inhibitors or apoptosis inducers to be developed as effective M.
tuberculosis therapeutics. These compounds would provide suitable starting points to further
develop these scaffolds using medicinal chemistry approach.
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Authors Contributions
RS planned the study and designed experiments. SK and TP performed microbiology and
animal experiments. RB and AB performed macrophage experiments. SC conducted the in
silico experiments and analyses. NK and CLM performed chemical synthesis. SD performed
autophosphorylation assays. RS, DM, AB, ME, DS, RD and NS analysed the results. RS and
SC wrote the manuscript with inputs from other authors.
Acknowledgments
The authors acknowledge the funding received from THSTI and Department of
Biotechnology, Govt. of India (BT/IN/Indo-Tunisia/01/2014). RS is a recipient of
Ramalingaswami fellowship and National Bioscience Award from Department of
Biotechnology. This research is also supported by Indian Institute of Science-Department of
Biotechnology partnership program to NS who is also a J. C. Bose National Fellow. Support
to NS for infrastructural facilities from Fund for Improvement of Science and Technology
infrastructure (FIST), DST and Centre for Advanced Structures (CAS), University Grants
Commission (UGC) is also acknowledged. Support to RD (EMR/2016/000048,
EEQ/2016/000205 and DST/INSPIRE/Faculty award/2014/DST/INSPIRE /04/2014/01662)
is acknowledged. The authors also thank Prof. R. Sowdhamini for her generous support in
providing access to Schrӧdinger suite of programs. We thank the technical staff of
Tuberculosis aerosol challenge facility, ICGEB for their help during animal experiments. RB
acknowledges Department of Science and Technology, India and FICCI for providing C.V.
Raman International fellowship. SC, TP and SM are thankful to DST-INSPIRE, DBT and
MHRD, Govt. of India for their respective fellowship. We acknowledge Dr. Sachchidanand
for useful discussions. Mr. Sushanth Pradhan (Biotechnology and Medical Engineering
department, NIT Rourkela) is gratefully acknowledged for technical assistance in confocal
microscopy experiment. The authors also acknowledge Anthem Biosciences, Bangalore for
pharmacokinetics studies. The technical help provided by Mr. Rajesh and Mr. Sher Singh is
highly acknowledged.
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Competing interests
The authors declare no competing interests
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