Inhibitory potential of some chalcones on cathepsins B, H and L

Article abstract of DOI:10.1039/c5ra12856k

Cathepsins, intracellular proteases, are known to be involved in a number of physiological processes such as degradation of extracellular proteins, prohormone processing, progressions of atherosclerosis etc. High levels of cathepsins have also been indica

Full text of DOI:10.1039/c5ra12856k

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INHIBITORY POTENTIAL OF SOME CHALCONES ON CATHEPSINS B, H AND L
SHWETA GARG and NEERA RAGHAV*
Department of Chemistry, Kurukshetra University, Kurukshetraꢀ136119 (INDIA)
Eꢀmail: nraghav.chem@gmail.com
Abstract
Cathepsins, intracellular proteases, are known to be involved in a number of physiological
processes ranging from degradation of extracellular proteins, prohormone processing,
progressions of atherosclerosis etc. High levels of cathepsins have also been indicated in various
pathological conditions like arthritis, cancer etc. One of the reasons for these elevated levels is
attributed to decrease in inhibitor concentration. Therefore, the work on the identification of
small molecular weight compounds as inhibitors of cysteine proteases is of great therapeutic
significance. In the present work, we report synthesis of a small library of chalcones and their
study as inhibitors to cysteine proteases. After a preliminary screening of the compounds as
inhibitors to cysteine proteases in general, studies were carried out to evaluate their inhibitory
effects on cathepsin B, H and L. The most potent inhibitors among all the compounds were nitro
substituted compounds for cathepsin B and cathepsin L and chloro substituted compounds for
cathepsin H, respectively.
Keywords: Chalcones, Cathepsin B, Cathepsin H, Cathepsin L, inhibitors
Introduction
Recent studies have shown that tumour growth, invasion and metastasis are strongly correlated
to the behaviour of cellular proteolytic enzymes [1]. Cellular proteases are generally subdivided
into four groups: serine, cysteine, aspartic and metalloproteinases. Cathepsins B, H and L (CB,

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CH and CL) lysosomal proteolytic enzymes, belonging to the cysteine protease family, widely
distributed in almost all mammalian cells are responsible for intracellular protein turnover [2]. It
has been demonstrated that cathepsins cannot only degrade components of the extracellular
matrix, such as laminin, collagen and elastin structures of basement membranes, but also
activate other proteolytic enzyme systems [3]. In vivo, the activity of cysteine proteases is
controlled by specific endogenous inhibitors, e.g. sterns and cystatins [4]. The catalytic
cathepsin activity is regulated by the balance between the amount of active enzyme and the
amount of the inhibitor. Imbalance between proteases and their inhibitors is believed to promote
tumour progression [5]. Recent studies have shown that cytosols of tumours tissues contain
higher levels of CB, CH, CL than normal adjacent tissue. Higher enzyme activity correlates
with tumour progression and shortened patient survival [6, 7]. This has been demonstrated in
lung, head and neck, colon and other cancers [7ꢀ 9].
Literature survey suggests that a large work has been done on peptidyl or peptidyl analogues as
inhibitors to cysteine proteases [10, 11]. However, these inhibitors are not considered to be
viable drug candidates for treating diseases like cancer, apoptosis etc. because of gastric
instability or the possibility of immunogenic reactions. Therefore, research on nonꢀpeptidyl
drugs has become an important aspect in drug research and development [12, 13]. Chalcone
derivatives (I and II) [14, 15] have been reported as inhibitors to cysteine proteases.
R1
R4
R9
Cl
Cl
R2
R3
R10
R11
R8
R7
O
Te
O
R6
Chalcone derivative (I)
4,4-dichloro-1,3-diphenyl-4-telluraoct-2-en-1-one(II)

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Toward this endeavor we have also studied the effect of some semicarbazones,
thiosemicarbazones, pyrazoles and pyrazolines as inhibitors of endogenous proteolytic activities
[16ꢀ21]. Some of the bischalcones and their derivatives [22], Acyl Hydrazides and Triazoles
[23], chalcone hydrazones [24] have been reported as effective nonꢀpeptidyl inhibitors for
cathepsin B & H from our laboratory. Chalcones constitute an important group of natural
products and have been reported to exhibit a wide variety of pharmacological effects including
antitumor activity [25], antimalarial [26], antiplatelet [27], antiviral [28], immunomodulatory
[29], antiꢀinflammatory [28 and anticancer agents [30]. These molecules are known to inhibit
various enzymes found responsible in the diseased conditions.
Previously we have reported that 2’ꢀhydroxychalcones are effective inhibitors of cathepsin B &
H [31]. In another study 4’ꢀphenylchalcones are also established as effective inhibitors of
cathepsins B, H and L [32]. To explore the work further and to establish a structureꢀ activity
relationship, a small library of chalcones with varying substituent having potential role as
antitumor and antiꢀinflammatory agents has ignited a thought to evaluate the designed
molecules as inhibitors to cysteine proteases, cathepsin B, H and L which may provide new
therapeutic opportunities in cancer treatment.
RESULT AND DISCUSSION
Synthesis of substituted 1, 3ꢀdiarylpropenones in quantitative yields by a quick solvent free
condensation method motivated us to synthesize a library of chalcones with varying substituent
(Scheme 1). During the process we observed that the yield was more and the process was fast,
the product was obtained in just 15 to 30 min. The resulting chalcones were identified with the
help of spectral studies. In the IR spectra of chalcones 1ꢀ110, the identification peak at 1651 ꢀ
1645 cmꢀ1 represented conjugated >C=O stretching vibrations, another characteristic peak at

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1605 ꢀ 1585 cmꢀ1 was attributed to C=C stretching vibrations. The results show the presence of
1
double bond in conjugation with carbonyl group in the synthesized compounds. HNMR
(CDCl3) spectrum, the Cꢀ2 and Cꢀ3 protons are observed as doublets with coupling constant ~
16 Hz. which shows that stereochemistry across Cꢀ2, Cꢀ3 double bond is Trans. The other
protons were revealed at their respective position as detailed in supplementary material.
Effect of synthesized compounds on in-vitro endogenous proteolysis in Liver homogenate
The effect of synthesized chalcones on proteolysis of endogenous protein substrates was observed at pH 5.0.
The proteolytic studies on endogenous protein substrates were conducted for 3h and 24h. Table
S1(supplementary data) presents the inhibition of endogenous proteolytic study in presence of different
compounds at pH 5.0 where most of the proteolytic activity is attributed to cysteine proteases [33]. It can be
observed that proteolytic activity is inhibited appreciably in presence of these compounds. In some
cases ~100% inhibition is achieved at 1×10ꢀ4M concentration. The results show that enzymatic
activities were influenced by the substituents on the ring and play a crucial role in inhibiting the
proteolytic activities. The enzyme activities vary from compound to compound depending on the
substituent present in all the synthesized compounds.
It may be mentioned here that the enzyme preparation contain many proteases. To establish a
general line of structure activity relationship is very difficult in this experiment but certainly some
trends can be visualized. One observation is that chalcones synthesized by the reaction of various
arylmethylketone with nitro substituted benzaldehyde from all the series show maximum inhibition.
The behavior might be attributed to its strong electron withdrawing nature. In most of the cases;
inhibition was more at 3h and less at 24h. The reason may be that binding of proteases with synthesized
compounds might have been reversed with time. In the initial stages, binding is more and results in high
inhibition and activity is regenerated with time resulting in lesser or no inhibition after 24h reaction
time. It may also be interpreted that with time some enzymes might have been activated which are less

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sensitive towards these synthesized compounds.
It can be observed from Table S1(supplementary data) that among various chalcones tested
maximum inhibition was observed in the case of 3ꢀ(3ꢀnitrophenyl)ꢀ1ꢀ(furanꢀ2ꢀyl)propꢀ2ꢀenꢀ1ꢀone
(1i) followed by 3ꢀ(3ꢀnitrophenyl)ꢀ1ꢀ(4ꢀhydroxyphenyl)propꢀ2ꢀenꢀ1ꢀone (5i), 3ꢀ(4ꢀnitrophenyl)ꢀ
1ꢀ(4ꢀ(N’ꢀ4ꢀbromophenyl)methylene)aminophenyl)propꢀ2ꢀenꢀ1ꢀone (11h), 3ꢀ(2ꢀnitrophenyl)ꢀ1ꢀ
(4ꢀaminophenyl)propꢀ2ꢀenꢀ1ꢀone (2h), 3ꢀ(3ꢀnitrophenyl)ꢀ1ꢀ(4ꢀfluorophenyl)propꢀ2ꢀenꢀ1ꢀone
(6i), 3ꢀ(3ꢀnitrophenyl)ꢀ1ꢀ(4ꢀbromophenyl)propꢀ2ꢀenꢀ1ꢀone (10i), 3ꢀ(4ꢀnitrophenyl)ꢀ1ꢀ(4ꢀ
(N’methylphenyl)propꢀ2ꢀenꢀ1ꢀone(7j), 3ꢀ(2ꢀnitrophenyl)ꢀ1ꢀ(pyridinꢀ2ꢀyl)ꢀ2ꢀenꢀ1ꢀone (9h), 3ꢀ(3ꢀ
nitrophenyl)ꢀ1ꢀ(4ꢀnitrophenyl)propꢀ2ꢀenꢀ1ꢀone (3i), 3ꢀ(2ꢀnitrophenyl)ꢀ1ꢀ(napthalenꢀ3ꢀyl)ꢀ2ꢀenꢀ
1ꢀone (8h), 3ꢀ(4ꢀnitrophenyl)ꢀ1ꢀ(thienꢀ2ꢀyl)propꢀ2ꢀenꢀ1ꢀone (4j) at 3h reaction. The same trend
was observed at 24h reaction but the extent of inhibition was decreased (Figure 2).
The data reported here indicate that these compounds act as inhibitors to proteases active at
pH 5.0, and inhibit endogenous protein hydrolysis significantly. In some cases ~100% inhibition is
ꢀ4
achieved at 1×10 M concentration. Once the inhibitory effects of compounds, series 1ꢀ11, was
established on cysteine proteases in general, their effect was observed on cathepsin B, H and L,
keeping in view the broad utility of these class of compounds as anticancer drugs as well as cysteine
protease inhibitor potentiality.
Effect of synthesized compounds on the activity of Cathepsin B
The activities of cathepsin B were estimated at varying concentrations of different categories of
synthesized compounds. Figure 3i show the relationship between the enzyme activity and
concentration of most inhibitory chalcone of each series (1ꢀ11), respectively. Among the various
compounds tested, it has been found that cathepsin B activity is inhibited and is affected by the
substituent present in compounds.
It was found that in chalcones from each series (1ꢀ11), 4ꢀnitrochalcone phenyl 3ꢀ(2ꢀnitrophenyl)ꢀ1ꢀ

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(furanꢀ2ꢀyl)propꢀ2ꢀenꢀ1ꢀone 1h showed maximum inhibition. Similarly, nitro substituted chalcone
in each series e.g. 3ꢀ(3ꢀnitrophenyl)ꢀ1ꢀ(4ꢀaminophenyl)propꢀ2ꢀenꢀ1ꢀone 2i, 3ꢀ(4ꢀnitrophenyl)ꢀ1ꢀ
(4ꢀnitrophenyl)propꢀ2ꢀenꢀ1ꢀone 3j, 3ꢀ(3ꢀnitrophenyl)ꢀ1ꢀ(thienꢀ2ꢀyl)propꢀ2ꢀenꢀ1ꢀone 4i, 3ꢀ(3ꢀ
nitrophenyl)ꢀ1ꢀ(4ꢀhydroxyphenyl)propꢀ2ꢀenꢀ1ꢀone
5i,
3ꢀ(3ꢀnitrophenyl)ꢀ1ꢀ(4ꢀ
fluorophenyl)propꢀ2ꢀenꢀ1ꢀone 6i, 3ꢀ(4ꢀnitrophenyl)ꢀ1ꢀ(4ꢀ(methylphenyl)propꢀ2ꢀenꢀ1ꢀone 7i, 3ꢀ
(3ꢀnitrophenyl)ꢀ1ꢀ(napthalenꢀ3ꢀyl)ꢀ2ꢀenꢀ1ꢀone 8h, 3ꢀ(3ꢀnitrophenyl)ꢀ1ꢀ(pyridinꢀ2ꢀyl)ꢀ2ꢀenꢀ1ꢀone
9i, 3ꢀ(2ꢀnitrophenyl)ꢀ1ꢀ(4ꢀbromophenyl)propꢀ2ꢀenꢀ1ꢀone 10h and 3ꢀ(4ꢀnitrophenyl)ꢀ1ꢀ(4ꢀ(N’ꢀ4ꢀ
methyl)methylene)aminophenyl)propꢀ2ꢀenꢀ1ꢀone 11j have been found to inhibit cathepsin B
appreciably.
.
Effect of synthesized compounds on the activity of Cathepsin H
The activities of cathepsin H were estimated at varying concentrations of different categories of
synthesized compounds. Figure 3ii show the relationship between the enzyme activity and
concentration of most inhibitory substituted chalcone of each series (1ꢀ11), respectively. Among the
various compounds tested, it has been found that cathepsin H activity is inhibited and is also
affected by the substituent present in compounds.
It was found that in chalcones from each series (1ꢀ11), 3ꢀ(3ꢀchlorophenyl)ꢀ1ꢀ(furanꢀ2ꢀyl)propꢀ2ꢀ
enꢀ1ꢀone 1c showed maximum inhibition. The compounds 3ꢀ(3ꢀchlorophenyl)ꢀ1ꢀ(4ꢀ
aminophenyl)propꢀ2ꢀenꢀ1ꢀone 2c, 3ꢀ(4ꢀchlorophenyl)ꢀ1ꢀ(4ꢀnitrophenyl)propꢀ2ꢀenꢀ1ꢀone 3d, 3ꢀ
(2ꢀchlorophenyl)ꢀ1ꢀ(thienꢀ2ꢀyl)propꢀ2ꢀenꢀ1ꢀone
hydroxyphenyl)propꢀ2ꢀenꢀ1ꢀone 5c, 3ꢀ(3ꢀchlorophenyl)ꢀ1ꢀ(4ꢀfluorophenyl)propꢀ2ꢀenꢀ1ꢀone 6c,
3ꢀ(4ꢀchlorophenyl)ꢀ1ꢀ(4ꢀ(N’methylphenyl)propꢀ2ꢀenꢀ1ꢀone 7d, 3ꢀ(2ꢀchlorophenyl)ꢀ1ꢀ
(napthalenꢀ3ꢀyl)ꢀ2ꢀenꢀ1ꢀone 8b, 3ꢀ(2ꢀchlorophenyl)ꢀ1ꢀ(pyridinꢀ2ꢀyl)ꢀ2ꢀenꢀ1ꢀone 9b, 3ꢀ(2ꢀ
chlorophenyl)ꢀ1ꢀ(4ꢀbromophenyl)propꢀ2ꢀenꢀ1ꢀone 10b, 3ꢀ(4ꢀchlorophenyl)ꢀ1ꢀ(4ꢀ(N’ꢀ4ꢀ
nitrophenyl)methylene)aminophenyl)propꢀ2ꢀenꢀ1ꢀone 11c also inhibited cathepsin H to a
4b,
3ꢀ(3ꢀchlorophenyl)ꢀ1ꢀ(4ꢀ

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greater extent when compared with other substituent in the series
.
Effect of synthesized compounds on the activity of Cathepsin L
The activities of cathepsin L were estimated at varying concentrations of different categories of
synthesized compounds. Figure 3iii show the relationship between the enzyme activity and
concentration of most inhibitory substituted chalcone of each series (1ꢀ11), respectively. Among the
various compounds tested, it has been found that cathepsin L activity is inhibited and is also affected
by the substituent present in compounds.
It was found that in chalcones from each series (1ꢀ11), 3ꢀ(2ꢀnitrophenyl)ꢀ1ꢀ(furanꢀ2ꢀyl)propꢀ2ꢀenꢀ
1ꢀone 1h showed maximum inhibition, followed by 3ꢀ(3ꢀnitrophenyl)ꢀ1ꢀ(4ꢀaminophenyl)propꢀ2ꢀ
enꢀ1ꢀone 2i, 3ꢀ(2ꢀnitrophenyl)ꢀ1ꢀ(4ꢀnitrophenyl)propꢀ2ꢀenꢀ1ꢀone 3h, 3ꢀ(3ꢀnitrophenyl)ꢀ1ꢀ(thienꢀ
2ꢀyl)propꢀ2ꢀenꢀ1ꢀone 4i, 3ꢀ(4ꢀnitrophenyl)ꢀ1ꢀ(4ꢀhydroxyphenyl)propꢀ2ꢀenꢀ1ꢀone 5j, 3ꢀ(3ꢀ
nitrophenyl)ꢀ1ꢀ(4ꢀfluorophenyl)propꢀ2ꢀenꢀ1ꢀone 6i, 3ꢀ(2ꢀnitrophenyl)ꢀ1ꢀ(4ꢀ(methylphenyl)propꢀ
2ꢀenꢀ1ꢀone 7h, 3ꢀ(4ꢀmethoxyphenyl)ꢀ1ꢀ(napthalenꢀ3ꢀyl)ꢀ2ꢀenꢀ1ꢀone 8g, 3ꢀ(3ꢀnitrophenyl)ꢀ1ꢀ
(pyridinꢀ2ꢀyl)ꢀ2ꢀenꢀ1ꢀone 9i, 3ꢀ(3ꢀnitrophenyl)ꢀ1ꢀ(4ꢀbromophenyl)propꢀ2ꢀenꢀ1ꢀone 10i, 3ꢀ(4ꢀ
nitrophenyl)ꢀ1ꢀ(4ꢀ(N’ꢀ4ꢀmethylphenyl)methylene)aminophenyl)propꢀ2ꢀenꢀ1ꢀone 11j in that
order
.
Enzyme kinetic studies
After establishing the inhibitory action of synthesized compounds on cathepsin B, cathepsin H
and cathepsin L, experiments were designed to evaluate the type of inhibition and to determine
the Ki value of these compounds on cathepsins B, H and L. For that, enzyme activities were
evaluated at different substrate concentrations in presence and absence of a fixed concentration
of different compounds. The enzyme concentration was kept constant in all the experiments.
Lineꢀweaver Burk plots drawn between 1/S and 1/V in presence and absence of most inhibitory
compound for cathepsin B, H and L are shown in figure 3iv, 3v and 3vi, respectively. It was

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found that the plots of 1/V and 1/S were straight lines intersecting at the Yꢀaxis and showed that
value of Vmax remained constant in all the compounds whereas the value of Km changes in
presence of each compound. These studies suggested a competitive type of inhibition exhibited
by these compounds for cathepsin B, cathepsin H and Cathepsin L. Using the Lineꢀweaver Burk
equation for competitive inhibition the Ki values were calculated and the Ki values for most
inhibitory compound of each series has been presented in table 1. The Ki value of most
inhibiting compound for cathepsin B in the each series has been evaluated as ~6.40x10ꢀ6M,
~6.20x10ꢀ6M, ~5.98x10ꢀ6M, ~6.57x10ꢀ6M, ~5.84x10ꢀ6M, ~5.94x10ꢀ6M, ~6.05x10ꢀ6M, ~6.12x10ꢀ
6M, ~5.91x10ꢀ6M, ~5.871x10ꢀ6M and ~5.81x10ꢀ6M for compounds 1h, 2i, 3j, 4i, 5i, 6i, 7i, 8h, 9i,
10h and 11j, respectively; Similarly for cathepsin H, the Ki value of most inhibiting compound
in the each series has been evaluated as ~297.8x10ꢀ6M, ~128.0x10ꢀ6M, ~405.4x10ꢀ6M,
~216.94x10ꢀ6M, ~163.3x10ꢀ6M, ~31.69x10ꢀ6M, ~41.6x10ꢀ6M, ~98.9x10ꢀ6M, ~47.24x10ꢀ6M,
~21.42x10ꢀ6M and ~13.3x10ꢀ6M for compounds 1c, 2c, 3d, 4b, 5c, 6c, 7d, 8b, 9b, 10b and 11c,
respectively. For cathepsin L, the Ki value of most inhibiting compound in the each series has
been evaluated as ~34.9x10ꢀ9M, ~19.4x10ꢀ9M, ~7.79x10ꢀ9M, ~72.9x10ꢀ9M, ~5.32x10ꢀ9M,
~6.41x10ꢀ9M, ~9.21x10ꢀ9M, ~10.66 x10ꢀ9M, ~14.48x10ꢀ9M, ~5.73x10ꢀ9M and ~4.4x10ꢀ9M for
compounds 1h, 2i, 3h, 4i, 5j, 6i, 7h, 8g, 9i, 10i and 11j, respectively.

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Tableꢀ1: Ki values exerted by most inhibitor compounds of each series on Cathepsin B, Cathepsin H and Cathepsin L
Cathepsin B
Ki
Cathepsin H
Cathepsin L
Code of
compound
S.No.
Code of
compound
Code of
Ki
Ki
(10ꢀ9M)
34.9
19.4
7.79
72.9
5.32
6.41
9.21
10.66
14.48
5.73
4.4
(10ꢀ6M)
6.40
6.20
5.98
6.57
5.84
5.94
6.05
6.12
5.91
5.871
5.81
compound
(10ꢀ6M)
297.8
128.0
405.4
216.94
163.3
31.69
41.6
1.
2.
3.
4.
5.
6.
7.
8.
9.
1h
2i
3j
4i
5i
6i
7i
8h
9i
10h
11j
1c
2c
3d
4b
5c
1h
2i
3h
4i
5j
6i
7h
8g
9i
10i
11j
6c
7d
8b
9b
10b
11c
98.9
47.24
21.42
13.3
10.
11.
Lineweaver – Burk plots were plotted between 1/V and 1/S to find the type of inhibition. It was found that all the compounds showed
competitive reversible inhibition on cathepsin B having specific activity 11.15 nmoles/min/mg, cathepsin H having specific activity
22.91nmoles/min/mg and cathepsin L having specific activity16.78 nmoles/min/mg (Fig 3iv-vi). The Ki values were calculated using
Lineweaver – Burk equations for competitive reversible inhibitions.
Tableꢀ2: Docking studies showing decrease in different energies of cathepsin B in presence of different chalcones (most inhibitor
chalcones from different series)
Compound
BANA
Total Energy
ꢀ129.75
VDW
ꢀ92.20
H Bond
ꢀ34.78
Electronic
ꢀ2.77
ꢀ106.89
ꢀ91.63
ꢀ92.81
ꢀ84.86
ꢀ84.65
ꢀ53.33
ꢀ71.54
ꢀ66.05
ꢀ22.23
ꢀ38.54
ꢀ21.5
0
Leupeptin
1h
0.23
0.23
0.32
2i
3j
ꢀ19.14

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4i
5i
ꢀ87.33
ꢀ96.49
ꢀ86.57
ꢀ83.26
ꢀ93.58
ꢀ88.07
ꢀ97.79
ꢀ100.66
ꢀ67.22
ꢀ74.39
ꢀ67.67
ꢀ71.71
ꢀ71.56
ꢀ65.65
ꢀ66.89
ꢀ88.54
ꢀ21.23
ꢀ22.33
ꢀ19.55
ꢀ11.79
ꢀ22.85
ꢀ23.05
ꢀ31.13
ꢀ12.12
1.12
0.23
0.65
0.24
0.82
0.63
0.23
0
6i
7i
8h
9i
10h
11j
The results are one of the docking experiments run using iGemdock under drug screening settings. The ligands were loaded as MDL
mol file. The active site was extracted from the structure of cathepsin B retrieved from Protein Data Bank ( http://www.rcsb.org/) as
cav2IPP B_PYS.pdb34.
Tableꢀ3: Docking studies showing decrease in different energies of cathepsin H
in presence of different chalcones (most inhibitor chalcones from different series)
Compound
Leuꢀ βNA
Total Energy
ꢀ75.71
VDW
ꢀ72.21
H
Bond
ꢀ3.5
Electronic
0
ꢀ61.28
ꢀ67.49
ꢀ72.52
ꢀ86.01
ꢀ64.34
ꢀ70.42
ꢀ44.86
ꢀ61.92
ꢀ62.05
ꢀ63.85
ꢀ64.34
ꢀ60.33
ꢀ16.41
ꢀ5.57
ꢀ10.47
ꢀ22.16
0
0
0
0
0
0
0
LeuCH2Cl
1c
2c
3d
4b
5c
ꢀ10.08

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6c
7d
ꢀ69.77
ꢀ68.67
ꢀ71.76
ꢀ66.84
ꢀ67.72
ꢀ98.57
ꢀ62.89
ꢀ68.67
ꢀ65.76
ꢀ56.34
ꢀ58.88
ꢀ89.07
ꢀ6.87
0
0
0
0
0
0
0
8b
ꢀ5.99
ꢀ10.5
ꢀ8.83
ꢀ9.5
9b
10b
11c
The results are one of the docking experiments run using iGemdock under drug screening settings. The ligands were loaded as MDL
mol file. The active site was extracted from the structure of cathepsin H retrieved from Protein Data Bank ( http://www.rcsb.org/) as
cav8PCH H_NAG.pdb35.
Tableꢀ4: Docking studies showing decrease in different energies of cathepsin L
in presence of different chalcones (most inhibitor chalscones from different series)
Total Energy
120.59
VDW
87.627
H Bond
ꢀ31.93
ꢀ31.06
ꢀ23.87
ꢀ16.5
Electronic
ꢀ1.724
2.290
00
Compound
Leupeptin
ꢀ136.04
ꢀ107.26
ꢀ68.150
ꢀ75.3242
ꢀ81.7823
ꢀ71.0995
ꢀ82.1074
ꢀ74.4905
ꢀ71.183
ZꢀPheꢀArgꢀ4mβNA
ꢀ92.022
1h
2i
ꢀ91.008
0.816
0
ꢀ87.8985
ꢀ88.216
ꢀ6.11
3h
4i
ꢀ18.34
ꢀ5.69
1.225
0
ꢀ87.806
5j
ꢀ93.6011
ꢀ93.4324
ꢀ19.91
ꢀ23.27
0.799
1.029
6i
7h

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ꢀ88.1839
ꢀ94.4156
ꢀ90.341
ꢀ81.2786
ꢀ79.1661
ꢀ70.9597
ꢀ95.229
ꢀ6.90
ꢀ15.25
ꢀ20
0
0
8g
9i
0.618
0
10i
11j
ꢀ102.184
ꢀ6.95
The results are one of the docking experiments run using iGemdock under drug screening settings. The ligands were loaded as MDL
mol file. The active site was extracted from the structure of cathepsin L retrieved from Protein Data Bank ( http://www.rcsb.org/) as
cav3BC3L_CSW36.

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Structure Activity Relationship Studies
In vitro inhibition studies of differently functionalized chalcones on cathepsins B, H and L
clearly demonstrated that the designed compounds inhibit these enzymes effectively. We have
demonstrated here that in general, these compounds exhibited an inhibition order cathepsin L>
> cathepsin B > cathepsin H. Establishing these compounds as potential inhibitors to cathepsins
B, H and L with targetꢀdirected chemical specificity suggests an optimistic future for their use
as cysteine protease inhibitors. These chalcones also provide a backbone skeleton for a number
of compounds to be used as therapeutic agents in a number of disease processes and are
important intermediates for various biologically active compounds. In order to ascertain
inhibition ability of the studied compounds, results were compared with Leupeptin, a known
inhibitor of cathepsins B and L and LeuꢀCH2ꢀCl for cathepsin H. It is reported that, Leupeptin is
a potential peptide inhibitor of cathepsin B [37], inhibited the goat brain cathepsin B [38]
competitively with Ki value of 12·5 × l0–9M whereas Ki value for human liver cathepsin Β [39]
was reported to be 7·0 × 10–9 M. In contrast, Ki value for human liver cathepsin H [39] was
reported to be 9.2 × 10–6 M. It has been reported that cathepsin L is inhibited by leupeptin with
a Ki value of 1.45 x 10ꢀ9 M [40]
Cl
O
O
H
H
O
N
N
NH2
NH
N
N
CH2
H
H
O
H N
H
2
Leu-CH2-Cl
Leupeptin
It can be observed that leupeptin showed ~ 98.5% inhibition at 10ꢀ6 M concentration for
cathepsin B. It showed ~ 51.89% inhibition at 10ꢀ5 M concentration for cathepsin H and ~
9.02% inhibition is observed for cathepsin L at 10ꢀ9 M concentration. Similarly, LeuꢀCH2ꢀCl

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showed ~ 10% inhibition at 10ꢀ5 M concentration for cathepsin B and 93.99% inhibition for
cathepsin L at 10ꢀ5 M concentration whereas it showed ~ 91.89% inhibition at 10ꢀ5M
concentration for cathepsin H. The results obtained are comparable with earlier results reported
for brain cathepsin H, cathepsin B and cathepsin L [38, 41,42].
The inhibitory potential of chalcones can be attributed to the presence of αꢀß unsaturated
carbonyl moiety in the molecule. Chalcones have previously been reported as inhibitors of
cathepsin B [43, 44]. The –CH2SH moiety present at the active site of enzymes can interact with
the electron deficient centre present in the chalcones inhibiting the enzymes activity. In the
present study, substitution pattern in each series was carefully altered to acquire a
comprehensible correlation between the electronic environment of ligands and the enzyme
active site or binding site. Among the similarly substituted designed compounds nitro
substituent has been evaluated as most inhibitory to enzymes cathepsin B and L. In a similar
study on bischalcones and quinazolineꢀ2(1H)ꢀone and quinazolineꢀ2(1H)ꢀthione derivatives
[22], we have evaluated that nitro substituted quinazolineꢀ2(1H)ꢀone and quinazolineꢀ2(1H)ꢀ
thione derivatives are potential inhibitors to cathepsins B and H. In another study, carried out in
order to identify non peptidyl inhibitors to cathepsins B and H, we have reported that 3ꢀphenylꢀ
5ꢀ(4ꢀnitrophenyl)ꢀ4ꢀaminoꢀ1,2,4ꢀtriazole and 3ꢀphenylꢀ5ꢀ(3’ꢀnirophenyl)ꢀ4ꢀaminoꢀ1,2,4ꢀtriazole
[23] inhibited cathepsins B and H effectively. In the present study, it can be observed that nitroꢀ
substitution provided an effective interaction for cathepsins B and L. All the synthesized
compounds were evaluated as better inhibitors to cathepsins B and L as compared to cathepsin
H. It may be an indication that active sites of cathepsins B and L are more susceptible to these
compounds as compared to cathepsin H. the results when compare with previously reported
inhibitory potential of 2’ꢀhydroxychalcones and their cyclized derivatives also suggest that –

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NO2 group plays an important role in effective binding of ligand with the enzyme active site.
The study reports that cathepsin B is strongly inhibited by nitro substituted chalcone with the Ki
value of 6.18x10ꢀ8 M [31]. In addition, it is further observed that 2’ꢀhydroxy group plays an
important role in the binding of chalcone with enzyme, as suggested by the Ki values reported
for 2’ꢀhydroxychalcones [31]. In case of Cathepsin H, we evaluated that halogen substituted
chalcones were most inhibitory. The results are similar to those reported previously [23] where
halogen substituted pyrazolines inhibited Cathepsin H appreciably. In the present study, we
have discussed that backbone structure as well as substituent effects the enzyme activities. And
found that in chalcones, ArꢀCOꢀCH=CHꢀAr’, substituents in the Ar’ affected the enzyme
inhibition to a significant extent. However, distinctive difference in inhibition pattern is not
observed when we changed the Ar group. The results are further discussed with this
background. The results were found consistent when compared with in- silico docking studies.
3.4. Molecular docking experiment
On the basis of the interaction data of docking experiments that include total energy and
individual energy terms, an indicative of the fitness of a predicted pose in the binding site, it is
suggested that the level of interaction is highest for 3ꢀ(4ꢀnitrophenyl)ꢀ1ꢀ(4ꢀ(N’ꢀ4ꢀ
methyl)methylene)aminophenyl)propꢀ2ꢀenꢀ1ꢀone 11j with in the active site of cathepsin B
(table 2) Fig 4 (iꢀxi) shows the result of the best docking pose of most inhibitory compounds of
each series 1h, 2i, 3j, 4i, 5i, 6i, 7i, 8h, 9i, 10h and 11j, respectively into the active site of
Cathepsin B. In cathepsin B, all the compounds showed a lesser interaction than the peptidyl
inhibitor, leupeptin. Decrease in total energy for leupeptinꢀcathepsin B has come out be ꢀ106.89
whereas with substrate BANA it has been computed as, ꢀ129.76 of which the contribution of
van der Waal interactions are more with a score of ꢀ84.66 as compared to Hꢀbonds with a score

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of ꢀ22.23. This is due to peptideꢀprotein interaction. Leupeptin is peptidyl in nature and
therefore binds effectively with the enzyme active site resulting in higher binding energy. As
compared to this the binding energy of title compounds are less (table 2). The in-silico
predictable behavior of enzymeꢀligand interaction can give an idea about the interaction.
Docking methods have been used to provide valuable insight into the binding mode between the
ligand and the enzyme active site thereby have an important role in the understanding of ligandꢀ
enzyme interactions. When compared with in the designed series to evaluate the interaction with
cathepsin B, the results clearly support the in vitro inhibition studies. In-vitro inhibition studies
(table S1)
(supplementary
data)
also show that 3ꢀ(4ꢀnitrophenyl)ꢀ1ꢀ(4ꢀ(N’ꢀ4ꢀ
methyl)methylene)aminophenyl) propꢀ2ꢀenꢀ1ꢀone 11j is the most potent inhibitor of cathepsin
B. It can be observed that Glnꢀ23, Gluꢀ28, Cysꢀ29, Trpꢀ30, Gluꢀ74 and Glyꢀ198 have been
found to interact with the substrate through Hꢀbonds. These amino acids interact with the
compounds under consideration; thereby suggest a competitive type of inhibition (Fig 3iv). The
proposed mechanism is shown in scheme 2. The results clearly indicate the significance of the
inꢀvitro inhibition studies.
In cathepsin H, the decrease in total energy with the reference inhibitor LeuꢀCH2Cl was less as
compared to all the designed compounds (Table 3). Here, it can be seen that though LeuꢀCH2Cl
is specific inhibitor for cathepsin H [42, 45], but possess only one amino acid residue as
compared to leupeptin–cathepsin B. therefore, the leuꢀCH Clꢀcathepsin H interaction cause a
2
decrease in energy of –61.282 of which ꢀ44.868 is the interaction and ꢀ16.414 is due to
hydrogen bonds. As listed in table 1, all the designed compounds have been found to show
higher decrease in ligand cathepsin H interaction energy than leuꢀCH2Clꢀ cathepsin H. In case
of cathepsin H, the level of interaction is highest for 3ꢀ(4ꢀchlorophenyl)ꢀ1ꢀ(4ꢀ(N’ꢀ4ꢀnitrophenyl)

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methylene) aminophenyl) propꢀ2ꢀenꢀ1ꢀone 11c with the active site of cathepsin H (table 3).
Figure 5 (iꢀxi) show the docked poses of the most inhibitory compounds of each series 1c, 2c,
3d, 4b, 5c, 6c, 7d, 8b, 9b, 10b and 11c into the binding site of Cathepsin H, respectively. As we
can see that the designed compound and the substrate leuꢀβNA binds at same site i.e. Serꢀ69,
Glnꢀ78 and Asnꢀ112 and this supports the results obtained during the inꢀvitro studies; the
compounds are evaluated as competitive inhibitors (Fig. 3v).
In case of cathepsin L, on the basis of the interaction data of docking experiments that include
total energy and individual energy terms, an indicative of the fitness of a predicted pose in the
binding site, it is suggested that the level of interaction is highest for 3ꢀ(4ꢀnitrophenyl)ꢀ1ꢀ(4ꢀ(N’ꢀ
4ꢀmethyl)methylene)aminophenyl)propꢀ2ꢀenꢀ1ꢀone 11j with the active site of cathepsin L (table
4). Fig 6 (iꢀxi) shows the result of the best docking pose of most inhibitory compounds of each
series i.e., 1h, 2i, 3h, 4i, 5j, 6i, 7h, 8g, 9i, 10i and 11j, respectively into the active site of
Cathepsin L. In cathepsin L, all the compounds showed a lesser interaction than the peptidyl
inhibitor, leupeptin. Decrease in total energy for leupeptinꢀcathepsin L has come out be ꢀ120.59
which is equivalent to substrate ZꢀPheꢀArgꢀ4mβNA, ꢀ136.05 of which the contribution of van
der Waal interactions are more with a score of ꢀ87.67 as compared to Hꢀbonds with a score of ꢀ
31.19. This difference can be explained on the basis of peptidyl nature of leupeptin as has been
explained for cathepsin L. As compared to this the binding energy of title compounds are less
(table 4). The amino acids Glnꢀ19, Trp ꢀ24 and Glyꢀ164 have been found to interact with the
compounds as well as with substrate ZꢀPheꢀArgꢀ4mβNA thus indicating a competitive type of
inhibition (Fig. 3vi) and the results clearly support the in vitro inhibition studies.. 3ꢀ(4ꢀ
nitrophenyl)ꢀ1ꢀ(4ꢀ(N’ꢀ4ꢀmethyl)methylene)aminophenyl) propꢀ2ꢀenꢀ1ꢀone 11j show maximum
inhibition (table 1) and therefore can account for maximum inhibition.

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In the present work decrease in total energy of enzyme –legand complex is presented as binding
affinity of ligand with in the active site of enzyme. However, Zhang et. al. have used free
energy based approach in understanding such interactions [46].
Experimental Section
All the chemicals (analytical grade) and biochemicals Fast Garnet GBC (oꢀaminoazotoluene
diazonium salt, substrate aꢀNꢀbenzoylꢀD, Lꢀarginineꢀ2ꢀnaphthylamide (BANA), LeuꢀßNA and
ZꢀPheꢀArgꢀ4mβNA were purchased either from Sigma Chemical Co., USA or from Bachem
Feinchemikalien AG, Switzerland. All the solutions used were prepared fresh in glassꢀdistilled
conductivity water. The protein sample was concentrated using Amicon stirred cells with YM
10 membrane under nitrogen pressure of 4–5 psi. The source of enzyme, fresh goat liver, was
obtained from local slaughter house.
General Procedure
Melting points were determined in open capillary tubes and are thus uncorrected. All the
chemicals and solvents used were of laboratory grade. IR spectra (KBr, cmꢀ1) were recorded on
1
a PerkinElmer spectrometer. H NMR spectra was recorded on Bruker 300 MHz NMR
spectrometer (chemical shifts in d ppm) using TMS as an internal standard. Thin layer
chromatography on aluminium plates percoated with silica gel G (Merck) in various solvent
systems using iodine vapors as detecting agent or by irradiation with ultraviolet lights (254 nm)
were used to monitor progress of reaction. ELISA plate reader was used for measuring
absorbance in the visible range. Refrigerated ultracentrifuge Remi Cꢀ24BL was used for
centrifugation purpose.
General method for the synthesis of 1, 3ꢀdiarylpropꢀ2ꢀenꢀ1ꢀones
1, 3ꢀdiarylpropꢀ2ꢀenꢀ1ꢀones were prepared under solvent free condensation of arylmethylketone
and arylaldehydes by grinding them with a mortar and pestle in the presence of solid sodium

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hydroxide. For this, equimolar ratio of arylmethylketone and potassium hydroxide pellets were
grinded in mortar with the help of pestle after making a paste, added equimolar ratio of aryl
aldehydes. The reaction was worked up in ice cold water. It was then filtered, washed with ice
cold water, dried and recrystallised from ethanol. The purity of compounds was checked with
the help of melting points, IR, 1H NMR and 13C NMR (supplementary material).
PROTEOLYTIC STUDIES
Preparation of liver homogenate: Goat liver, purchased fresh from the local slaughter house
was washed with cold isotonic saline solution. The tissue was then homogenized in 0.1M
acetate buffer pH 5.5 containing 0.2M NaCl in a mixerꢀcumꢀblender to obtain 10% (w/v)
homogenate and was stored at 4oC.
Assay for proteolytic activity: The proteolytic activity was estimated at pH 5.0, 37ₒC using 0.1
M acetate buffer as the incubation medium. The homogenate prepared above was incubated
with the buffer at 37oC for 3 h and 24 h., separately. The reaction was stopped by the addition of
TCA and the resulting solution was centrifuged to precipitate proteins. The acid soluble proteins
were quantitated in the supernatant using Bradford method [47]. The experiments were
conducted in triplicate and the results are presented in table S1(supplementary data) (figure 1 and
figure 2).
Purification of goat brain cathepsin Β, cathepsin H and cathepsin L
All the purification steps were carried out at 4°C. Cathepsin B, H and L were isolated, separated
and purified from goat liver using the following procedure [48]. Goat liver acetone powder
homogenisation in cold 0.1 M sodium acetate buffer pH 5.5 containing 0.2 M NaCl and 1mM
EDTA, Acidꢀautolysis by lowering pH from 5·5 to 4·0 by gradual addition of cold 1 Ν HCl, 30ꢀ
80% ammonium sulphate fractionation. Fractionation of proteases based on molecular weight

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on Sephadex Gꢀ100 column chromatography and finally cationꢀexchange chromatography on
CMꢀSephadex Cꢀ50 and DEAE Sephadex Aꢀ50 column. The specific activities of the cathepsin
B, cathepsin H and cathepsin L were ~11.15 nmoles/min/mg, ~22.91 nmoles/min/mg and
~16.78 nmoles/min/mg, respectively.
Effect of compounds on the activity of Cathepsin B
Cathepsin Β was determined spectrophotometrically using BANA as a substrate at pH 6.0. The
activities of cathepsin B was estimated at varying concentrations of synthesized compounds
(table S1 (supplementary data) shows activities of cathepsin B at minimum inhibitory
concentration of synthesized compounds). First of all, enzyme was equilibrated in phosphate
buffer of pH 6.0 at 37oC. The stock solutions of compounds were prepared in DMSO. Then
appropriate amount of individual compounds were added in the reaction mixture separately to
effect the final concentration of each compound as 0.1, 0.5 and 1.0 X 10ꢀ4 M final
concentration. After an incubation time of 30 min. residual enzyme activity was estimated by
the usual enzyme assay [22] at pH 6.0 using αꢀNꢀbenzoylꢀD, Lꢀarginineꢀ2ꢀnaphthylamide
(BANA) as substrate. The experiments were performed in triplicate for each concentration and
averaged before further calculations. The % activity in each case has been calculated with
respect to the control where no compound was added but an equivalent amount of solvent was
present. The activities of cathepsin B estimated at varying concentrations of most inhibitory
compounds of each series have been shown in figure 3i.
Effect of compounds on the activity of Cathepsin H
Cathepsin H was determined spectrophotometrically using LeuꢀβNA as a substrate having
specific activity 22.91 nmoles/min/mg. The activities of cathepsin H was estimated at varying
concentrations of synthesized compounds (table S1 (supplementary data) shows activities of

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cathepsin H at maximum inhibitory concentration of synthesized compounds). First of all,
enzyme was equilibrated in phosphate buffer of pH 7.0 at 37oC. The stock solutions of
compounds were prepared in DMSO. Then appropriate amount of individual compounds were
added in the reaction mixture separately to effect the final concentration of each compound as
0.1, 0.5 and 1.0 X 10ꢀ3 M final concentration. After an incubation time of 30 min. residual
enzyme activity was estimated by the usual enzyme assay [22] at pH 7.0 using LeuꢀβNA as
substrate. The experiments were performed in triplicate for each concentration and averaged
before further calculations. The % activity in each case has been calculated with respect to the
control where no compound was added but an equivalent amount of solvent was present. The
activities of cathepsin H estimated at varying concentrations of most inhibitory compounds of
all series have been shown in figure 3ii.
Effect of compounds on the activity of Cathepsin L
Cathepsin L was determined spectrophotometrically using ZꢀPheꢀArgꢀ4ꢀmβNA as a substrate
having specific activity 16.78 nmoles/min/mg. The activities of cathepsin H was estimated at
varying concentrations of synthesized compounds (table S1 (supplementary data) shows activities
of cathepsin L at maximum inhibitory concentration of synthesized compounds). First of all,
enzyme was equilibrated in phosphate buffer of pH 6.0 at 37oC. The stock solutions of
compounds were prepared in DMSO. Then appropriate amount of individual compounds were
added in the reaction mixture separately to effect the final concentration of each compound as
0.1, 0.5 and 1.0 X 10ꢀ6 M final concentration. After an incubation time of 30 min. residual
enzyme activity was estimated by the usual enzyme assay [38] at pH 6.0 using ZꢀPheꢀArgꢀ
4mβNA as substrate. The experiments were performed in triplicate for each concentration and
averaged before further calculations. The % activity in each case has been calculated with

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respect to the control where no compound was added but an equivalent amount of solvent was
present. The activities of cathepsin L estimated at varying concentrations of most inhibitory
compounds of all series have been shown in figure 3iii.
Kinetic studies of synthesized compounds on cathepsin B, cathepsin H and cathepsin L
After establishing the inhibitory action of synthesized compounds on cathepsin B, cathepsin H
and cathepsin L; experiments were designed to evaluate the type of inhibition and to determine
the Ki value of these compounds on respective enzymes. For that, enzyme activities were
evaluated at different substrate concentrations in presence and absence of a fixed concentration
of inhibitor. The enzyme concentration was kept constant in all the experiments. Lineꢀweaver
Burk plot were drawn between 1/S and 1/V in presence and absence of compounds on cathepsin
B, cathepsin H and cathepsin L (Figure 3 iv, v, vi). The Ki values were calculated using the
Lineꢀweaver Burk equation for competitive inhibition Km’= Km(1+I/ Ki).
DRUG MODELING STUDIES
All docking studies were performed using iGemdock. For these studies, small molecular weight
ligands and enzyme active site structure is required. The structure of cathepsin B [34], cathepsin
H and cathepsin L were retrieved from Protein Data Bank as (cav2IPP B_PYS.pdb), and
(cav8PCH H_NAG.pdb) [35] and cav3BC3L_CSW [36], respectively. The structures were
prepared in Marvin sketch, minimized and were saved as MDL Mol File. The prepared ligands
and the binding site were loaded in the iGemdock software and docking was started by setting
the GAꢀ parameters at drug screening setting. The results are presented in table 2, 3 and 4
pertain to the interaction data. Fitness is the total energy of a predicted pose in the binding site.
The empirical scoring function of iGemdock is the sum total of Van der Waal, Hꢀbonding and
electrostatic energy. The docked poses of the ligands in the active site of cathepsin B, H and L

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along with their respective substrates and most inhibitory compounds of each series are shown
in figure 4,5 and 6.
CONCLUSION
In summary, we have demonstrated that these compounds showed more inhibition on cathepsin
L activity in comparison to cathepsin B and H. Analysis of the effect of these compounds on
Cathepsin B, H and L by the combination of serendipitous biologic selectivity with targetꢀ
directed chemical specificity suggests an optimistic future for their use as cysteine protease
inhibitors as therapy in a number of disease processes.
ACKNOWLEDGEMENT
The authors are thankful to Department of Science and Technology, New Delhi for funding the
project. One of the authors, Shweta Garg is thankful to and UGC New Delhi, India for award of
SRF and also to Kurukshetra University, Kurukshetra for providing necessary research
laboratory facilities. The authors have declared no conflict of interest.
REFERENCES
1. C. C. Calkins and B. F. Sloane, Biol Chem Hoppe Seyler, 1995, 376, 71ꢀ80.
2. Y. Uchiyama, S. Waguri, N. Sato, T. Watanabe, K. Ishido and E. Kominami, Acta
Histochem Cytochem, 1994, 27, 287ꢀ308.
3. M. R. Buck, D. G. Karustis, N. A. Day, K. V. Honn and B. F. Sloane, Biochem J, 1992,
282, 273ꢀ278.
4. B. Turk, J. G. Bieth, I. Bjork, I. Dolenc, D. Turk, N. Cimerman, J. Kos, A. Colic, V.
Stoka and V. Turk, Biol Chem Hoppe Seyler, 1995, 376, 225ꢀ230.
5. Y. M. Henskens, E. C. Veerman and A. V. Nieuw Amerongen, Biol Chem Hoppe
Seyler, 1996, 377, 71ꢀ86.

RSC Advances
Page 24 of 34
View Article Online
DOI: 10.1039/C5RA12856K
6. L. Benitez Bribiesca, G. Martinez, M. T. Ruiz, F. Gutierrez Delgado and D. Utrera, Arch
Med Res, 1995, 26, S163ꢀS168.
7. E. Campo, J. Munoz and R. Miquel, et al., Am J Pathol 1994, 145, 301ꢀ309.
8. W. Ebert, H. Knoch, B. Werle, G. Trefz, T. Muley and E. Spies, Anticancer Res, 1994,
14, 895ꢀ899.
9. M. Budihna, P. Strojan and L. Smid, et al., Biol Chem Hoppe Seyler, 1996, 377, 385ꢀ
390.
10. H. H. Otto and T. Schirmeister, Chem. Rev., 1997, 97, 133ꢀ171.
11. D. Steverding, The Open Enzyme Inhibition Journal, 2011, 4, 11ꢀ16. V. Turk, J. Kos,
and B. Turk, Cancer Cell, 2004, 5, 409ꢀ410.
12. D. Dana., A. R. Davalos, S. De, P. Rathod, R. K. Gamage, J. Huestis, N. Afzal, Y.
Zavlanov, S. S. Paroly, S. A. Rotenberg, G. Subramaniam, K. J. Mark, E. J. Chang and
S. Kumar, Bioorg. and Med. Chem., 2011, 21(11), 2975ꢀ2987.
13. P. Schenker, P. Alfarano, P. kolb, A. Caflisch and A. Baici, Protein Science, 2008, 17,
2145ꢀ2155.
14. I. Caracelli, M. VegaꢀTeijido, J. ZukermanꢀSchpector, M. H. S. Cezari, J. G. S. Lopes,
L. Juliano, P. S. Santos, J. V. Comasseto, R. L. O. R. Cunha and E. R. T. Tiekink, J of
Molecular Struct., 2012, 1013, 11ꢀ18.
15. R. Li, G. L. Keynon, F. E. Cohen, X. Chen, B. Gong, J. N. Dominguez, E. Davidson, G.
Kurzban, R. E. Miller, E. Q. Nuzum, P. J. Rosenthal and J. H. McKerrow, J Med.
Chem., 1995, 38, 5031ꢀ5037.
16. N. Raghav, R. Kaur, M. Singh, Suman and Priyanka, Asian J. Chem., 2010, 22, 7097ꢀ
7101.

Page 25 of 34
RSC Advances
View Article Online
DOI: 10.1039/C5RA12856K
17. N. Raghav, M. Singh, R. Kaur, Suman and Priyanka, J. Pharma Tech., 2010, 2, 743ꢀ
749.
18. N. Raghav, M. Singh, R. Kaur, Suman and Priyanka, Asian J. Chem., 2011, 23, 1409ꢀ
1410.
19. R. Kaur, M. Singh, S. Jangra and N. Raghav, Int. J. Chem. Sci., 2012, 10, 1698ꢀ1704.
20. M. Singh and N. Raghav, Int. J. Pharmacy and pharmaceut. Sci., 2013, 5, 80ꢀ86.
21. M. Singh and N. Raghav, Int. J. Pharmacy and pharmaceut. Sci., 2013, 5, 365ꢀ368.
22. N. Raghav and M. Singh, Eur. J. Pharma. Sci., 2014, 54, 28ꢀ39.
23. N. Raghav and M. Singh, Eur. J. Med. Chem., 2014, 77, 231ꢀ242.
24. Raghav, N., Singh, M., 2014c. SAR studies of differently functionalized chalcones
based hydrazones and their cyclized derivatives as inhibitors of mammalian cathepsin B
and cathepsin H. Bioorg. Med. Chem. DOI:10.1016/j.bmc.2014.05.037.
25. S. Mukherjee, V. Kumar, A. K. Prasad, H. G. Raj, M. E. Bracke, C. E. Olsen, S. C. Jain
and V. S. Parmar, Bioorg. Med. Chem., 2001, 9, 337–345.
26. T. Narender, Shweta, K. Tanvir, M. S. Rao, K. Srivastava and S. K. Puri, Bioorg. Med.
Chem. Lett., 2005, 15, 2453.
27. M. Zhao, H. S. Jin, L. P. Sun, H. Piao and Z. S. Quan, Bioorg. Med. Chem. Lett., 2005,
15, 5027.
28. S. Cheenpracha, C. Karapai, C. Ponglimanont, S. Subhadhirasakul and S. Tewtrakal,
Bioorg. Med. Chem., 2006, 14, 1710.
29. L. Barford, K. Kemp, M. Hansen and A. Kharazmi, Int. Immunopharmacol, 2002, 2,
545ꢀ550.

RSC Advances
Page 26 of 34
View Article Online
DOI: 10.1039/C5RA12856K
30. A
.
Modzelewska, C. Pettit, G. Achanta, N.E. Davidson, P. Huang and S. R. Khana,
2006, 14(10), 3491
Bioorg Med Chem
,
.
31. Raghav, N., Garg, S. 2014. SAR studies of oꢀhydroxychalcones and their cyclised
analogs and study them as novel inhibitors of Cathepsin B and Cathepsin H. European
Journal of Pharmaceutical Sci. 60, 55ꢀ63.
32. Raghav, N.; Ravish, I. RSC Adv. 2015, 5, 50440ꢀ53.
33. N. Raghav, M. Singh, R. Kaur, Suman and Priyanka, Asian J. Chem., 2011, 23, 1409ꢀ
1410.
34. C. P. Huber, R. L. Campbell, S. Hasnain, T. Hirama and R. To, Crystal structure of the
tetragonal form of human liver cathepsin B. (http://www.ebi.ac.uk/pdbeꢀ
srv/view/entry/2ipp/citation.html) (31st 1 May 2013).
35. G. Guncar, M. Podobnik, J. Pungercar, B. Strukelj, V. Turk, D. Turk, Structure, 1998, 6,
51–61.
36. S. F. Chowdhary, L. Joseph, S. Kumar, S. R. Tulsidas, S. Bhat, E. Ziomek, R. M.
Menard, J. Sivaraman and E. O. Purisima, J. Med. Chem., 2008, 51, 1361ꢀ1368.
37. A. Baici and M. GygerꢀMarazzi, Eur. J. Biochem., 1982, 129, 33ꢀ41.
38. R. C. Kamboj, S. Pal and H. Singh, J. Biosci., 1990, 15, 397–408.
39. C. G. Knight, Biochem. J., 1980, 189, 447ꢀ453.
40. A. Azaryan and A. Galoyan, Neurochem. Res., 1987, 12, 207ꢀ213.
41. R. C. Kamboj, S. Pal, N. Raghav, H. Singh, Biochimie, 1993, 75, 873ꢀ878.
42. N. Raghav, R. C. Kamboj, S. Parnami and H. Singh, Indian J Biochem. Biophys., 1995,
32, 279ꢀ85.
43. S. H. Kim, E. Lee, K. H. Baek, H. B. Kwon, H. Woo, E. S. Lee, Y. Kwon, Y. Na,
Bioorg. and Med. Chem. Letters, 2013, 23, 3320ꢀ3324.

Page 27 of 34
RSC Advances
View Article Online
DOI: 10.1039/C5RA12856K
44. I. Caracelli, M. VegaꢀTeijido, J. ZukermanꢀSchpector, M. H. S. Cezari, J. G. S. Lopes,
L. Juliano, P. S. Santos, J.V. Comasseto, R. L. O. R. Cunha and E. R. T. Tiekink, J. of
Molecular Struct., 2012, 1013, 11ꢀ18.
45. W. N. Schwartz, A. J. Barrett, Biochem. J, 1980, 191, 487ꢀ497.
46. Z. Zhang, V. Martiny, D. Lagorce, Y. Ikeguchi, E. Alexov and M. A. Miteva, PLoS
One, 2014, 9, e1108884.
47. M. M. Bradford, Anal. Biochem., 1976, 72, 248ꢀ254. C. M. Stoschek, Enzymol, 1990,
18, 250ꢀ68.
48. N. Raghav, M. Singh, S. Garg, I. Ravish, R. Kaur and Suman, Int. J. pharmaceut. sci.
res. 6(7), (2015) 2944ꢀ2949.
Figure Captions
Figure 1: Effect of chalcones on the endogenous proteolytic activity for 3 hr reaction. The
data in each bar represents the % Residual Activity in presence of individual compound
w.r.t. control taken as 100.
Figureꢀ2: Effect of compounds on the endogenous proteolytic activity 24 hr reaction. The
data in each bar represents the % Residual Activity in presence of individual compound
w.r.t. control taken as 100.
Figure 3: Effect of varying concentration of most inhibitory chalcones from each series,
1h, 2i, 3j, 4i, 5i, 6i, 7i, 8h, 9i, 10h and 11j on cathepsin B figure 3(i) , 1c, 2c, 3d, 4b, 5c, 6c,
7d, 8b, 9b, 10b and 11c, on cathepsin H figure 3(ii) and 1h, 2i, 3h, 4i, 5j, 6i, 7h, 8g, 9i, 10i,
11j, on cathepsin L activity 3(iii), respectively. Results are mean of experiments conducted
in triplicate. % Residual activities are presented w.r.t control which contain equivalent
amount of solvent.
Line ꢀ weaver Burk plot for cathepsin B at varying concentrations of BANA in presence of
1x10ꢀ5M concentration of most inhibitory chalcone of each series (3iv), at pH 6.0. The Km
value for control have been found to be 3.64x10ꢀ4 M. Line ꢀ weaver burk plot for cathepsin
H on varying concentrations of leuꢀßNA in presence of 1x10ꢀ4M concentration of most
inhibitory chalcone of each series (3v), at pH 7.0. The Km value for control has been

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found to be 5.34x10ꢀ4M. Lineꢀweaver Burk plot for cathepsin L at varying concentrations
of ZꢀPheꢀArgꢀ4mβNA in presence of 1x10ꢀ7M concentration of most inhibitory chalcone of
each series (3vi), at pH 6.0. The Km value for control has been found to be 6.024x10ꢀ5 M.
The Ki values as calculated from this graph are presented in table 1.
Figure 4: Docking results showing the alignment of most inhibitory compounds along with
the BANA in the active site of cathepsin B (cav2IPP B_PYS.pdb). Here fig. 4(iꢀxi) show
alignment of 1h, 2i, 3j, 4i, 5i, 6i, 7i, 8h, 9i, 10h and 11j along with BANA in the active site
of cathepsin B (cav2IPP B_PYS.pdb), respectively. Active site amino acid Cysꢀ29 interacts
with all the compounds as well as with BANA through Hꢀbonds explaining the competitive
type of inhibition (Fig 3iv).
Figure 5: Docking results showing the alignment of most inhibitory compounds along with
the LeußNA in the active site of cathepsin H (cav8PCH H_NAG.pdb) Here fig. 5(iꢀxi) show
alignment of 1c, 2c, 3d, 4b, 5c, 6c, 7d, 8b, 9b, 10b and 11c, along with LeußNA in the active
site of cathepsin H (cav8PCH H_NAG.pdb), respectively. Serꢀ69, Glnꢀ78 and Asnꢀ112
interact with the designed compound and the substrate leuꢀβNA as well through Hꢀbonds
suggesting a competitive type of inhibition (Fig 3v).
Figure 6: Docking results showing the alignment of most inhibitory compounds along with
the zꢀPheꢀArgꢀ4mβNA in the active site of cathepsin L (cav3BC3L_CSW.pdb). Here fig.
6(iꢀxi) show alignment of 1h, 2i, 3h, 4i, 5j, 6i, 7h, 8g, 9i, 10i, 11j, along with ZꢀPheꢀArgꢀ
4mβNA in the active site of cathepsin L (cav3BC3L_CSW.pdb), respectively. The amino
acids Glnꢀ19, Trp ꢀ24 and Glyꢀ164 have been found to interact with the compounds as well
as with substrate ZꢀPheꢀArgꢀ4mβNA through Hꢀbonds thus indicating a competitive type
of inhibition (Fig 3vi).

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