Novel peptidomimetic peptide deformylase (PDF) inhibitors of Mycobacterium tuberculosis

Article abstract of DOI:10.1111/cbdd.13769

Emergence of MDR-TB and XDR-TB led to the failure of available anti-tubercular drugs. In order to explore, identify and develop new anti-tubercular drugs, novel peptidomimetic series of Mtb–peptide deformylase (PDF) inhibitors was designed and synthesized. In vitro antimycobacterial potential of compounds was established by screening of compounds against Mycobacterium tuberculosis H37Rv strain using MABA. Among them, ester series of compounds 4a, 4b, 4c, 4d, and 4e were found most active, with compound 4c being highly active and exhibiting minimum inhibitory concentration of 6.25?μg/ml against M.?tb H37Rv strain. Additionally, the compounds were docked to determine the probable binding interactions and understand the mechanism of action of most active molecules on Mtb-peptide deformylase (PDF), which is involved in the mycobacterium protein synthesis.

Full text of DOI:10.1111/cbdd.13769

DR VIKAS TELVEKAR (Orcid ID : 0000-0002-2074-1186)
Article type : Research Article
Novel peptidomimetic peptide deformylase (PDF) inhibitors of Mycobacterium tuberculosis
a
a*
Kunal M. Gokhale , Vikas N. Telvekar
aDepartment of Pharmaceutical Sciences and Technology, Institute of Chemical Technology,
Matunga, Mumbai-400 019, India
*corresponding author
Email Id: vikastelvekar@rediffmail.com
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
1
0.1111/CBDD.13769
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Abstract
Emergence of MDR and XDR-TB led to the failure of available anti-tubercular drugs. In-order to
explore, identify and develop new antitubercular drugs, novel peptidomimetic series of Mtb-
Peptide Deformylase (PDF) inhibitors was designed and synthesized. In-vitro antimycobacterial
potential of compounds was established by screening of compounds against Mycobacterium
tuberculosis H37Rv strain using MABA assay. Among them ester series of compounds 4a, 4b, 4c,
4d, 4e were found most active, with compound 4c being highly active and exhibiting minimum
inhibitory concentration of 6.25 µg/ml against M.tb H37Rv strain. Additionally; the compounds
were docked to determine the probable binding interactions and understand the mechanism of
action of most active molecules on Mtb-peptide deformylase (PDF), which is involved in the
mycobacterium protein synthesis.
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Keywords: Peptidomimetic, Mtb-Peptide Deformylase (PDF) inhibitor, MABA assay, docking,
Mycobacterium tuberculosis
1. Introduction
Peptide deformylase (PDF; EC 3.5.1.31) is an enzyme involved in bacterial protein synthesis
responsible for catalyzing formylation/deformylation steps in almost all bacteria including
Mycobacterium tuberculosis (Mtb). Its uniqueness lies in the fact that it is present in human
pathogen and absent in human cells, also it is part of an essential pathway of pathogen, easy to
assay in-vivo and in-vitro. Further, if inactivated it does not result in resistance or bypass process if
inactivated (Clements et al., 2001). Thus, PDF satisfies all the criteria listed for the attractive
bacterial target to offer next generation antibacterial drugs (Gigilone et al., 2000; Johnson et.al.,
2
005;Verma et al., 2011; Jain et al., 2005). Many of the marketed antibacterial and especially
antimycobacterial drugs target protein synthesis steps. This provides the basis to choose PDF as a
potential anti-tb target. The unexploited Mtb-PDF and its essentiality in bacterial protein synthesis
attracted us to develop a new class of potential anti-tb compounds. Peptide deformylase is an
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2
essential metalloprotease utilizing nickel (Ni ) at its active site. It catalyzes removal of N-formyl
group from substrate methionine (Mazel et al., 1997) (Figure 1).
X-ray structure of Mtb-PDF-inhibitor complex makes us to understand the structural requirements
for enzyme. Mtb-PDF has 3 binding pockets in active sites viz. S1’, S2’, S3’ (Aubart et al., 2007).
S1’ is the binding site for methionine side chain of substrate and composed of highly hydrophobic
residues. The active site of Mtb-PDF is highly conserved and particularly present in S1’ pocket
(Smith et al., 2003).
The S2’ pocket is not a true ‘pocket’ but rather an open tunnel like space pointing out into solvent
and able to accommodate wide variety of side chains presented by various formylated substrates
(Hu et al, 1999). S3’ pocket is least conserved region of active site between Gram-negative and
Gram-positive PDFs, due to a two-residue insertion between S1’ and S2’ regions in Gram-positive
species. This area is solvent exposed and more likely a surface depression than a true binding
pocket (Smith et al., 2003). Thus, inhibitors that target specific residues within this pocket are
considered to be more selective for those particular PDF (Aubart et al., 2007). Based on this,
following generalizations can be made about structural pattern of inhibitors and their binding
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pattern: P1: This group should be linear aliphatic chain and provide a kind of steric shield to the
molecule, P2: Presence of t-butyl group at this position shows best activity. Presence of isopropyl,
prolyl also showed activity, P3: Physicochemical, Pharmacokinetic properties of molecule are
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2
decided by this substitution (Verma et al., 2011). Ni is found to be present at the metal binding
site (MBG) of Mtb-PDF which is in coordination with three protein residues namely Cys-106,
His-148, His-152 (Pichota et al., 2008).
Although, bacterial PDF was identified 45 years ago, until 2008 X-ray crystallographic structure
of Mtb-PDF was not available (Adams, 1968), which led to difficulty in assay development,
screening and subsequent reports of variety of PDF inhibitors (Yuan et al., 2006). Actinonin was
very first bacterial PDF inhibitor of natural origin but could not reach to market due to poor
structural stability and quick clearance tendency (Broughton et al., 1975). LBM-415, GSK-
1
322322 were the candidates in phase I and II clinical trials respectively but suffered from major
toxicity issues like methemoglobinemia and generation of reactive toxic metabolites (Rolan et al.,
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2
011; Hoover et al., 2016; Naderer et al., 2013; Clinical Trial Study Reg. No. NCT01818011,
011). The very first report of PDF inhibitor against infections caused by Mycobacterium
tuberculosis (Mtb) dates back to 2006 and call our attention to LBK-611 and its analogs with
potentially new anti-mycobacterial agents (Teo et al., 2006) (Figure 2).
Studies on Mtb-PDF inhibitor complex indicate the presence of valine like side chains at P2 and
hydroxamic acid residues at Metal binding site. Replacement of any other amino acid at P2
decreases the inhibitory potential. It was thought to be due to absence of hydrogen binding of -NH
hydrogen with nearby water molecule or carbonyl oxygen in enzyme backbone (Lee et al., 2010).
This clearly marks the presence of H O molecule at active site. Reports are available which
2
indicate the requirement of acidic group for better binding interactions at metal binding site of
enzyme and inhibitor. It was observed that Ni+2 form square pyramidal co-ordination with
bidentate hydroxamic acid moiety. Hydroxamates are found to be the best groups for metal
binding site in comparison with reverse hydroxamic acids; carboxylic acid group has a negative
impact on inhibitory potential. With reference to the above discussion different functionalities
responsible for binding of actinonin at active site of PDF are shown in Figure 2. We also wish to
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2
report here the structure of an inhibitor co-crystallized with Mtb-Ni -PDF complex (PDB ID:
E3U) obtained from the protein data bank (Pichota et al., 2008). We have used this molecule as
3
native ligand for our study (Figure 2).
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Literature survey provides Comparative Molecular Field Analysis (COMFA) study for PDF
inhibitors and proposes three novel PDF inhibitors for Mtb-PDF (Vaidya et al., 2011) (Figure 2).
The structural features, significance and development took place in field of PDF inhibitors
prompted us to undertake design, synthesis, in-silico studies and biological screening of novel
PDF inhibitors against Mtb (Figure 2).
To the best of our knowledge, there have been no reports on Mtb-PDF inhibitors having ester or
hydrazide at metal binding site and aromatic or heteroaromatic ring at P3 site. We undertook a
comprehensive structure activity relationship (SAR) study and synthesis of Mtb-PDF inhibitors
equipped with ester or hydrazide at metal binding site and aromatic (substituted/unsubstituted) or
heteroaromatic rings at P3 site. With reference to literature we decided to maintain L-valine at P2
site in all analogues.
2
. Materials and Methods
All the reagents and substrates used were in their commercially available form and were used as
received and purchased from Rarco Research Lab, Mumbai, India. Selenium Dioxide was
obtained as gift sample from Omkar Chemicals, Mumbai, India. Pyruvic acid was purchased from
S. D. Fine chemicals limited, India. All the solvents were used as received without further
purification. Isoniazid, Rifampicin, Streptomycin sulphate and Ciprofloxacin hydrochloride were
obtained as gift samples from Calyx Chemicals and Pharmaceuticals Ltd., Mumbai and used as
received. All the reactions were monitored by thin-layer chromatography on silica gel plates (GF
2
54) and visualized with UV light. Melting points of the compounds were determined on DBK
Prog. melting point apparatus and were uncorrected. Specific optical rotation was measured using
Accumax Polarimeter. IR spectra were recorded in KBr pellets on Shimadzu IR Affinity-1 FTIR
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spectrophotometer. H NMR, C NMR spectra were recorded in CDCl , DMSO-d6, D O (if
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2
applicable) on a Bruker Avance II 400 MHz spectrometer with tetramethylsilane (TMS) as an
internal reference at SAIF, Panjab University, Chandigarh, India. The chemical shifts are given in
δ (ppm) referenced to the respective solvent peak and coupling constants are reported in Hz. Mass
spectra were recorded on Agilent MSD at NMIMS University, Mumbai, India. Almost all
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compounds were characterized by FTIR, H NMR, C NMR and HRMS.
2
.1 General procedures for preparation of substituted aromatic oxo acetic acids 2a-2j
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In a single neck 25 ml round bottom flask equipped with magnetic stirrer arylmethyl ketone (1a-1j,
eq.) and selenium dioxide (1.5 eq.) were added followed by anhydrous dry pyridine (10 mL).
1
o
o
The reaction mixture was heated at 110 C for 1hr. and bath temperature was then reduced to 90 C.
o
The reaction mixture was heated for 4 hrs. at 90 C. After completion of reaction (TLC), reaction
mixture was filtered residue was washed with ethyl acetate (2 x 50 mL). The combined filtrate was
treated with 1N HCl (20 mL) and organic layer was separated, washed with water, dried on
Na SO and concentrated on rotary evaporator. The pure glyoxylic acids 2a-j (76%-54%) were
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obtained by column chromatography using Ethyl acetate: n-Hexane (90:10) and characterized by
melting points and FTIR similar to reported compounds. (Wadhwa et al., 2008; Hatanaka et al.,
1
973; Jerchel et al., 1955)
2
.2 General procedure for preparation of L-valine ethyl ester hydrochloride 3
To a stirred suspension of L-Valine (1 eq.) in anhydrous ethanol (1.5ml) cooled in an ice bath,
thionyl chloride (1.9 eq.) was added dropwise. The reaction mixture was refluxed for 4 hrs. After
cooling equal quantity of toluene (150 ml) was added and all volatiles were removed using rotary
evaporator to yield solid L-valine ethyl ester hydrochloride. (Karanfil et al., 2012)
2
.3 General procedure for preparation of compound 4a-4j
o
To a cold solution (-5 C) of L-valine ethyl ester hydrochloride 3, (1 eq.) and triethylamine (1 eq.)
in acetonitrile (10 mL) was added compound (2a-j), dicyclohexylcarbodimide (1 eq.),
o
hydroxybenzotriazole (1 eq.) successively. The reaction mixture was stirred at 0 C for 2 hrs., then
o
at 5 C at 1 hr. and at room temperature for 8 hrs. The reaction mixture was set aside overnight.
The precipitated dicyclohexyl urea was filtered off and solvent was evaporated using rotary
evaporator. The resulted residue was dissolved in CHCl (30 mL) and further washed with 10%
3
HCl (3 X 30 mL) and water. The organic layer was dried over Na SO and evaporated using rotary
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4
evaporator to obtained crude product, which was purified by using silica gel column
chromatography using n-hexane: ethyl acetate (80:20) as eluting solvent. The compounds were
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characterized by melting point, H NMR, C NMR and mass spectrometer (Figure S7-S36).
2
.4 General procedures for preparation of compounds 5a-j
To a solution of 4a-j (1 eq.) in ethanol (10 mL) was added hydrazine hydrate (10 eq.). The mixture
was refluxed for 5 hrs. After completion of reaction (TLC), the reaction mixture was concentrated
using rotary evaporator and purified using silica gel column chromatography to get pure 5a-5j
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using n-hexane: ethyl acetate (80:20) as eluting solvent. The compounds were characterized by
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melting point, H NMR, C NMR and mass spectrometer (Figure S37-S76).
2
.5 Anti-tubercular evaluation
The anti-mycobacterial activity of compounds was assessed against M. tuberculosis using
Microplate Alamar Blue assay (MABA). This methodology is non-toxic, uses a thermally stable
reagent and shows good correlation with proportional and BACTEC radiometric method. Briefly,
2
00μl of sterile deionized water was added to all outer perimeter wells of sterile 96 wells plate to
minimize evaporation of medium in the test wells during incubation. The 96 wells plate received
00 μl of the Middlebrook 7H9 broth and serial dilution of compounds (4a-4j & 5a-5j) were made
1
directly on plate. The final drug concentrations tested were 100 to 0.2 μg/ml. Plates were covered
and sealed with parafilm and incubated at 37ºC for five days. After this time, 25μl of freshly
prepared 1:1 mixture of Almar Blue reagent and 10% tween 80 was added to the plate and
incubated for 24 hrs. A blue color in the well was interpreted as no bacterial growth, and pink
color was scored as growth. The MIC was defined as lowest drug concentration which prevented
the color change from blue to pink. Isoniazid, Rifampicin, Streptomycin Sulphate, Ciprofloxacin
Hydrochloride were used as standards for the study.
Standard Strain used: Mycobacterium tuberculosis (Vaccine strain, H37Rv strain): ATCC No-
2
7294.
2
.6 Docking studies and In silico ADME prediction
The docking studies were performed using Glide module (version 7.1, Schrödinger, LLC, NY)
installed on Linux workstation and In Silico ADME was predicted by Qikprop module as
described in the supplementary information.
2
.7 In vitro cytotoxicity study
The cytotoxicity of all novel 20 compounds (4a-4j & 5a-5j) was tested in THP-1differentiated
macrophages using MTT assay. Cells were allowed to adhere for 24 h in a 96-well plate at a
density of 3000 cells/well prior to the assay. Further, cells were exposed to various concentrations
of 0.9% saline (the control), compounds (4a-4j & 5a-5j) at various concentrations (125-6.25
o
µg/ml) for 48 h at 37 C and 5CO atmosphere, respectively. Culture medium was used as the
2
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blank group. After that, the MTT solution was added to each well and cells were incubated for 4 h.
To dissolve the MTT formazan crystals, 200 ml of DMSO was added to each well. A microplate
reader (Model 680, BIO-RAD, Hercules, CA) was used to measure the optical density of the
formazan product at 570 nm. The drug concentration causing 50% inhibition (IC50) was
calculated. Isoniazid, Rifampicin, Streptomycin Sulphate, Ciprofloxacin Hydrochloride were used
as standards for the study.
3
3
. Results and discussion
.1 Chemistry
The synthesis of novel peptidomimetic PDF inhibitors containing ester and hydrazide is depicted
in Scheme 1. Compound 2a-j were prepared by oxidation of substituted or unsubstituted aromatic
or heteroaromatic methyl ketones using SeO and pyridine. Compound 2d was obtained using
2
literature reported procedure involving oxidation of corresponding N-acetyl derivative with SeO2
in pyridine followed by hydrolysis with 6N HCl (Domagala et al., 1981). L-valine ethyl ester
hydrochloride 3 was obtained by literature reported procedure using thionyl chloride and ethanol.
The corresponding ethyl esters 4a-4j were obtained by reacting corresponding glyoxylic acids 2a-
2
j with L-valine ethyl ester hydrochloride 3 in presence of DCC, TEA, HOBT in acetonitrile as
solvent. For the synthesis of hydrazides 5a-5j corresponding ethyl ester 4a-4j was refluxed with
hydroxylamine hydrochloride (99%) in ethanol for 5 hrs. (Scheme 1). The structures of 4a-j and
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1
5
a-j were characterized by H NMR, C NMR, and mass spectroscopic analysis. In H NMR
spectra, the aromatic protons resonated at δ 7.35-9.50 ppm. Resonance signals of the aliphatic
region were seen in the range of δ 0.89 - 2.30 ppm. The ethyl ester was seen at δ 4.22-4.73 ppm.
1
3
Doublet between δ 8.20 - 8.48 ppm confirmed the presence of proton of -CONH group. In C
NMR spectra, aromatic carbon resonated between δ 126 - 149 ppm with many overlaps in these
regions, while the ketone C=O resonated around δ 180-185 ppm, amide C=O at about δ 161-165
ppm, ester C=O at around δ 170-172 ppm. The ethyl side chain appeared at δ 14-15 ppm and at δ
6
0-61 ppm whereas ispropyl appeared downfield at around δ 17-19 ppm. In case of hydrazide
series of compounds (5a-j) broad singlet at δ 9.0-9.5 ppm confirms the presence of –CONH group
1
and –NHNH protons resonated at δ 8.0-8.6 ppm. Disappearance of these signals in H NMR
2
using D O solvent validated the presence of –CONH, -NHNH in the synthesized compounds. In
2
2
13C NMR hydrazide C=O reasonated at δ 134-135 ppm, alongwith amide C=O at δ 163-165 ppm
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and ketone C=O appears downfield in comparison to corresponding ester series of compound at δ
70-172 ppm. Further structural confirmation was done using mass spectrometric data analysis
Supplementary Information).
1
(
3
.2 Biological Screening
Compounds were tested using Microplate Alamar Blue Assay (MABA) method using H37Rv
ATCC 27294) strain of Mycobatcerium tuberculosis by the standard procedure mentioned.
Strepromycin sulphate (STR), Isoniazid (INH), Rifampicin (RIF) and Ciprofloxacin hydrochloride
CIP) were used as standard to explore the potential of synthesized compounds against MDR-TB
(
(
and XDR-TB. All the synthesized compounds were evaluated in a whole-cell assay against Mtb
strain H37Rv using MABA assay. The synthesized compounds exhibited interesting activity with
MIC ranging from 100 µg/ml - 6.25 µg/ml against H37Rv as seen in Table 1. The results of the
antimycobacterial activity revealed that nature and type of aromatic ring is important for anti-
tubercular activity, also difference in activity depends upon substitution on aromatic ring.
Unsubstituted ester 4a and compounds 4b, 4c, 4d, 4e have shown comparably good anti-tubercular
activity, presence of electron releasing group like methoxy (4b) at para position of phenyl ring
gives equal activity as compared to unsubstituted counterpart. Amidst, compounds with electron
withdrawing groups (4c, 4e) at para position of phenyl ring; 4c shows the higher activity which
can be attributed to higher lipophilicity, H-bonding capacity and size of the substituent.
Introduction of strongly electron releasing group at para position of phenyl ring (4d) maintains the
activity as compared to unsubstituted counterpart. This underlines the need of electron
withdrawing substituent at para position of phenyl ring. Isosteric replacement of phenyl ring with
thiophene ring (4f) and furyl ring (4h) give compounds with equal potency. Replacements of
thiophene ring with 5-bromo-2-thiophenyl (4g), 3-pyridyl (4i) are detrimental for potency of
compounds. The resulting compounds are less potent and potency varies by the order 2-thiophenyl
=
2-furyl > 5-bromo-2-thiophenyl > 3-pyridyl, where compound with 2-thiophenyl and furyl refers
to more potent and 3-pyridyl is least potent. We also tested the influence of replacing aliphatic
side chain for phenyl ring on activity but the compound 4j was found to be resistant at all
concentrations ranging from 100 µg/ml – 0.8 µg/ml as per our expectation. This underlines the
role of lipophilicity, H-bonding capacity, type and size of substituent on aromatic ring at P3 site of
Mtb-PDF.
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+
2
In order to assess the metal binding capacity of compounds at Ni containing active site hydrazide
series of compounds 5a-5j was also synthesized but all compounds were found to be less potent as
compared to ester series of compounds. Consistent with pattern of activity as in case of ester series
of compounds, hydrazide series of compounds follow the order where unsubstituted (5a) is less
potent than para substituted electron withdrawing compound (5c). Both 5c, 5e are equipotent.
Introduction of electron releasing groups (5b, 5d) on the para position of aromatic ring maintains
the activity. Rather these compounds are equally active this may be attributed to very little change
in lipophilicity, H-bonding capacity of electron releasing group containing compounds of
hydrazide series. Isosteric replacement of phenyl ring with thiophene ring (5f) gives equally active
compound whereas activity decreases with introduction of 3-pyridyl ring; rather compound was
found to be resistant in MABA assay. Thus, type, size and electronic nature of para substituent on
aromatic ring is critical in activity of both ester and hydrazide series of compound, being 4c
(Figure 3) is highly potent compound of the series.
3
.3 Molecular Docking studies
Mtb-PDF is distinct from other bacterial PDF. So inhibition of Mtb-PDF is an ideal target. We are
reporting a modelling study of synthesized novel PDF inhibitors effective against Mtb-PDF. These
studies can provide useful information about the binding interactions at active site of Mtb-PDF.
Our study includes structure-based design approach docking study using Glide (version 7.1,
Schrödinger, LLC, NY). Ligand preparation was accomplished on all synthesized molecules using
+
2
Ligprep module (Version 2.1; Schrödinger, LLC, NY). A reported crystal structure of Mtb-Ni -
PDF in a complex with inhibitor (PDB ID: 3E3U) was obtained from protein data bank and
performed docking study. Docking procedures were used to identify the correct confirmation of
ligand in binding pocket of enzyme and to predict interaction between ligand and enzyme as seen
in Figure S1-S6, Table S1.
3
.4 In-Silico ADME prediction
To further explore the behaviour of synthesized series of compounds we have performed ADME
prediction studies by Qikprop. For ester series of compounds 4a-4j, the partition coefficient (QP
log O/W) was found to be in the range of 1.8 – 3.07 resembling to native ligand. Caco-2 cell
permeability (QPPCaco), a major factor to determine drug metabolism and its access to biological
cell membrane ranged between 99.454 – 849.836 nm/sec, from which most active molecule 4c
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shows an absorption value of 849.836 nm/sec, more than actinonin and native ligand. Overall
human oral absorption for 4a-4j is 73.522% – 100%. All these pharmacokinetic parameters were
found in acceptable limit defined for human use, thereby indicating its potential as drug (Table
S2). All the molecules of hydrazide series 5a-5j were found to be less hydrophobic as exhibited by
their partition coefficient (QP log O/W) value in the range of -0.389 – 1.121. Caco-2 cell
permeability of all these molecules was also found to be very less (Table S2) in the range of
1
8.293-153.127, which is attributed to presence of –CONHNH group that may undergo metabolic
2
degradation at faster rate. Also % human oral absorption of these compounds was very less.
Therefore, it can be concluded that high hydrophilicity, tendency to undergo metabolic
degradation and low human absorption are the reasons behind low activity of these compounds in
MABA assay, more specifically it is attributed to hydrophilicity because of known fact that
lipophilicity of molecule could ease the entry of molecule through lipid-enriched M.Tb cell
membrane (Biava et al., 2008; Navarrete-Vázquez et al., 2007).
3
.5. In vitro cytotoxicity study
From the therapeutic view, it is crucial that drugs should solely kill intracellular pathogen without
displaying a cytotoxic effect on the host cell. In the present study, the cytotoxic study of the
synthesized compounds was determined on THP-1 differentiated macrophages using MTT assay.
It was found that the synthesized compounds 4a-4j and 5a-5j had no cytotoxic effects on the
THP-1 macrophages. All the compounds were having IC50 value more than 125 µg/ml indicating
that the compounds were nontoxic upto 125 µg/ml. (Fig. 4).
4
. Conclusion
In conclusion, a novel peptidomimetic series of Mtb-PDF inhibitors were designed successfully.
These compounds were synthesized from readily accessible reactants and reagents by simple and
efficient synthetic protocols. Extensive SAR study of ester containing PDF inhibitors led us to
discover 4c, which then displays a good in-vitro anti-tubercular activity with MIC 6.25 µg/ml
against H37Rv (ATCC 27294) strain of Mtb. In-vitro cytotoxicity assay also revealed that
compounds are safe. This also supports that results obtained after in-vitro antimycobacterial
activity are intrinsic and solely due to the structures and no systemic toxicity has been observed.
Additionally, docking and MM-GBSA studies on the enzyme Mtb-PDF were carried out with the
most active molecules to examine the putative interactions responsible for biological activity.
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Presence of electron withdrawing substituent on aromatic ring, its size, H-bonding with
corresponding amino acids at active site and π-π interactions with His 148 are the reasons
attributed to high activity of 4c and moderate activity of 4b and 4e. Modelling study showed that
P2 and P3 side chains of 4c are nicely overlapped with native ligand. Our results showed that
peptidomimetic substituted aromatic ring containing ester scaffold can be used as promising class
of Mtb-PDF inhibitors. Further studies are underway to improve antitubercular potency and to
elucidate the detailed mechanism of action.
Acknowledgments
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Authors would like to thank SAIF, Panjab University for recording H and C NMR. Authors also
thank NMIMS University, Mumbai for HRMS analysis. Authors are also thankful to Maratha
Mandal’s Central Research Laboratory, Belgaum, India for providing antitubercular activity.
Conflict of interest
The authors declare no conflict of interest.
Figure Legends:
Figure 1. Schematic representation of formylation/deformylation cycle catalyzed by PDF
Figure 2. Reported and proposed PDF Inhibitors
Figure 3. Structure of most active molecule 4c
Figure 4. Results of In-vitro cytotoxicity by MTT assay. Each point represents the mean ± SD of
triplicate measurements
Scheme Legends:
Scheme 1. Synthesis of novel peptidomimetic PDF inhibitors 4a-4j and 5a-5j
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Supplementary Information
Additional supplementary information including figure, table, and spectra may be found online in
the supplementary information tab for this article.
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MAP
PDF
(
Methionine aminopeptidase)
N-formylmethionylpeptide
N-methionylpeptide
N-Peptide
PDF
Inhibitor
H O
H O Formate
2
Methionine
2
Inhibition
No Removal of Formyl group
Protein Synthesis Blocked
Mature Protein
Figure 1.
Schematic representation of formylation/deformylation cycle catalyzed by PDF
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a) Structures of PDF inhibitors
Actinonin
CH3
OH
N
O
O
O
O
N
H
N
HN
N
HO
O
N
N
HN
OH
H
O
O
CH3
O
NH
O
H3C
OH
O
H
LBK 611
N
F
N
HO
N
CH3
H
O
O
N
N
b) Reported co-crystallized native ligand
inhibitor of MTb-Ni+2-PDF
O
H
N
N
O
LBM 415
N
H
HO
N
N
H
N
O
O
F
N
GSK1322322
c) Reported PDF Inhibitors by COMFA analysis
OH
O
O
OH
O
NH
O
NH
NHOH
NHOH
CH3
O
NH
NHOH
OH
H C
CH3
3
H C
3
O
H C
CH3
3
Compound II
Compound I
Compound III
O
Metal binding group
R = - COOEt, - CONHNH2
d)Binding of Actinononin at active site of PDF
H
Physicochemical/
Pharmacokinetic
parameter P3
N
R
P1
CH3
Ar
O
H C
CH3
3
Metal Binding Site
OH
O
O
Steric Bulk
P3
Physicochemical
Pharmacolinetic
HO
NH
N
e) General structure of novel Mtb-PDF
inhibitors under study.
H
O
H C
CH3
3
P2
Figure 2. Reported and proposed PDF Inhibitors
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O
O
NH
OCH CH3
2
O
O N
2
H C
3
CH3
Figure 3. Structure of most active molecule 4c
1
1
20
00
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
5a
1
00
5
5
5
5
5
5
5
b
c
9
8
7
0
0
0
8
6
4
2
0
0
0
0
0
d
e
f
60
50
40
g
h
i
g
h
3
2
1
0
0
0
0
5i
j
STR
INH
RIF
CIP
STR
INH
RIF
0
50
100
150
Concentration (ug/ml)
0
100
200
CIP
Concentration (ug/ml)
(a)
(b)
Figure 4 Results of In-vitro cytotoxicity by MTT assay using THP-1 cells differentiated to
macrophages (a)(4a-4j) (b)5a-5j. Each point represents the mean ± SD of triplicate measurements.
INH: isoniazid, STR: Streptomycin, RIF: Rifampin, CIP: Ciprofloxacin.
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O
O
a
CH3
O
b
OH
L-Valine
Ar
H C
3
O
CH3
Ar
CH3
a-j
O
NH . HCl
2
.
.
1
2
a-2j
c
3
O
O
H
N
Ar
OCH CH3
2
O
H C
CH3
3
4a-4j
d
O
O
H
N
Ar
NHNH2
CH3
a-5j
O
H C
3
5
Scheme 1. Synthesis of novel peptidomimetic PDF inhibitors 4a-4j and 5a-5j
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Table 1. Anitubercular activity using Microplate Alamar Blue Assay (MABA)
O
O
H
N
R
R1
CH3
O
H C
3
Code No.
R
R1
MIC value (μg/ml)
4
a
Ph
COOC H
12.5
12.5
6.25
12.5
12.5
12.5
25
2
5
5
5
5
5
5
5
5
5
5
4
b
4-OMePh
COOC H
2
4
c
4-NO Ph
COOC H
2
2
4
d
4-NH Ph
COOC H
2
2
4
e
4-ClPh
COOC H
2
4
f
2-thiophenyl
COOC H
2
4
g
5-bromo-2-thiophenyl
COOC H
2
4
h
2-furyl
3-pyridyl
Me
COOC H
12.5
100
2
4
i
COOC H
2
4
j
COOC H
Resistant*
25
2
5
a
Ph
CONHNH2
CONHNH2
CONHNH2
CONHNH2
CONHNH2
CONHNH2
CONHNH2
CONHNH2
CONHNH2
CONHNH2
--
5
b
4-OMePh
25
5
c
4-NO Ph
12.5
25
2
5
d
4-NH Ph
2
5
e
4-ClPh
12.5
25
5
f
2-thiophenyl
5
g
5-bromo-2-thiophenyl
37.5
100
5
h
2-furyl
5
i
3-pyridyl
Resistant*
Resistant*
6.25
0.8
5
j
Methyl
Streptomycin
Rifampin
--
--
--
--
--
Isoniazid
--
12.5
1.6
Ciprofloxacin
--
*
Resistant: Not active at all concentrations ranging from 100 μg/ml – 0.8 μg/ml in MABA assay
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