Cathepsin B: Novel cysteine proteases of the papain family

Mr. L. Jayashankar

Cysteine proteinases of the papain superfamily are widely distributed in nature; they can be found in both prokaryotes and eukaryotes, e.g. bacteria, parasites, plants, invertebrates and vertebrates. In mammals, the major cysteine proteinases are the lysosomal cathepsins B and L.

They are implicated in many physiological processes such as protein degradation, antigen presentation and bone resorption but they also have been implicated in a number of degradative and invasive processes such as arthritis, tumor invasion and metastasis and muscular dystrophy.

Cathepsins are usually characterized as members of the lysosomal cysteine protease (active site) family and the cathepsin family name has been synonymous with lysosomal proteolytic enzymes. In actuality, the cathepsin family also contains members of the serine protease (cathepsin A, G) and aspartic protease (cathepsin D, E) families as well. These enzymes exist in their processed form as disulfide-linked heavy and light chain subunits with molecular weights ranging from 20-35 kDa. Cathepsin C is the noted exception, existing as an oligomeric enzyme with a molecular weight 200kDa. Cathepsins are initially synthesized as inactive zymogens; they are post-translationally processed into their active configurations after passing through the endoplasmic reticulum and subsequent incorporation into the acidic environment of the lysosomes.

Cathepsins are ubiquitous lysosomal proteases that are classified according to their active site. Structural differences between various cathepsins result in variations in their substrate specificity and mechanism of inhibition. Cathepsins play an important role in the turnover of intracellular proteins and extracellular proteins via endocytosis. Extracellularly they have been implicated in tumor invasion and metastasis and, recently, as a positive mediator of apoptosis induced by gamma-interferon, Fas/APO-1, and TNF-alpha.

Human Cathepsin B (1GMY) 

A-chain of Human Cathepsin B

The crystal structure of Human liver Cathepsin B (1GMY) has a resolution of 1.9 Å. It consists of Chains A, B and C with 339 residues. Cathepsin B has the following chemical components in its crystal structure: Diphenylacetic acid (DFA), 3-Methylphenylalanines (APD) and 2-Aminoethanimidic acid.

Physiological role

Lysosomal proteases and particularly the thiol proteinases, appear to be involved in the degradation of intracellular proteins in vivo. One of the best characterized of these enzymes is cathepsin B (Barrett, 1973) (EC 3.4.22.1).

Cathepsin B has well-established endopeptidase activity against proteins such as haemoglobin, casein, cartilage proteoglycan, collagen, myofibrillar proteins and immunoglobulin G. Cathepsin B inactivates three intracellular enzymes, glucokinase, pyruvate kinase and fructose-bisphosphate aldolase.

Cathepsin B acts as a peptidyldipeptidase with aldolase as substrate, i.e., there is a sequential cleavage of dipeptides from the C-terminus of the aldolase subunit. Cathepsin B cleaves up to nine dipeptides from the C-terminus and does not readily degrade the native protein further. Cathepsin B also degrades glucagon by a peptidyldipeptidase, indicates that this type of limited degradation may be an important general aspect of the action of cathepsin B on protein substrates.

Cathepsin B is an intracellular lysosomal cysteine protease primarily exhibiting carboxydipeptidyl activity. It is an abundant and ubiquitously expressed cathepsin. Together with other cathepsins, it is involved in the degradation and processing of proteins, including the regulation of pro-hormone and pro-enzyme activation, antigen processing, inflammatory responses against antigens, metabolism, tissue remodeling and apoptosis. Cathepsin B from the granule-derived cell surface has been suggested to provide self-protection for degranulating cytotoxic lymphocytes . There is evidence for its involvement in the activation of a plasminogen activator.

Apart from the lysosomal activity of cathepsin B, its extracellular activity in the lumen of thyroid follicles is a prerequisite for the solubilization of thyroglobulin and the liberation of thyroid hormones. Therefore, the secretion of lysosomal enzymes from within thyroid epithelial cells is of relevance for proper thyroid function.

Active site of Cathepsin B

Cysteine proteases contain a thiolate-imidazolium ion pair, the thiolate moiety of which acts as a nucleophile in attacking the substrate carbonyl carbon atom to form an acylenzyme intermediate. Unlike papain, cathepsin B is not only an endopeptidase but also an exopeptidase. Specifically, it can remove dipeptide units from the C termini of proteins and peptides. Comparison between the amino acid sequences of papain and cathepsin B has suggested that substitution of arginine for Ala-162 of papain can account for the exopeptidase activity of cathepsin B .

This peptidyldipeptidase activity is due to a structural feature particular to cathepsin B (when compared with other cysteine proteases), which is an extra peptide segment termed the occluding loop located in the primed side of the active site. On the basis of X-ray crystallographic studies, it was postulated that the exopeptidase activity is dependent on two histidine residues (His ¹¹⁰ and His ¹¹¹ ) located within the occluding loop and in the S2’ subsite. Two saltbridge interactions between the loop and the main body of the mature enzyme (Asp ²² His ¹¹⁰ and Arg ¹¹⁶ Asp ²²⁴ ) maintain it in a closed position, so that these histidine residues form the outer boundary of the S2 subsite. In contrast, the occluding loop adopts a drastically divergent conformation in procathepsin B where the propeptide passes directly through the active-site cleft, demonstrating the potential for mobility of this structural element in the active protease.

It is positioned within the active site cleft above the primed binding sites. At the bottom of the loop, there are two positively charged residues His ¹¹⁰ and His ¹¹¹ , which are responsible for the binding of the free C-terminal carboxy group of a substrate. Without knowing the structure of the enzyme, cathepsin B-selective inhibitors CA030 [EtO-(2 S ,3 S )- t Eps-Ile-Pro-OH] and CA074 [ n PrHN-(2 S ,3 S )- t Eps-Ile-Pro-OH] were developed, which were derived from E-64 [ trans -epoxysuccinyl-L-leucylamido-(4-guanidino)butane], a non-specific inhibitor of papain-like cysteine proteases.

Representation of the occluding loop region in the human cathepsin B

The role of the occluding loop has also been clarified by mutational analysis. It was demonstrated that deletion of the entire loop respectively eliminated exopeptidase activity while peptidyldipeptidase conserving endopeptidase activity. Removal of the Asp ²² His ¹¹⁰ and Arg ¹¹⁶ - Asp ²²⁴ ion pairs resulted in a dramatic increase in the endopeptidase activity of cathepsin B, as measured by extended quenched fluorescence substrates, suggesting that the loop is detected when peptides bind for cleavage in the endopeptidase mode. In addition, the pH dependency of the binding of the cathepsin B propeptide was shown to be saltbridge attributable to the Asp ²² - His ¹¹⁰ ion pair.

Pathological role

The ability of tumor cells to invade into the extracellular matrix has been attributed to the activity of cathepsins released by tumor cells or associated with the plasma membrane of tumor cells. Benign tumors are characterized by a continuous basal lamina separating the epithelium from the stroma. However, invasive carcinomas exhibit a disrupted extracellular lamina adjacent to the invading tumor cells in the stroma. Cathepsins secreted by invading tumor cells can degrade collagen and elastin, thereby destroying the basal laminar region. In normal cells, following their synthesis, cathepsins are transported into the lysosomal compartment. However, in tumor cells, instead of being transported into the lysosomal compartment, they are secreted into the surrounding medium. The presence of cathepsins in the extracellular compartment may be employed as an ideal independent prognostic factor to determine the clinical outcome of cancer chemotherapy.

Elevated Cathepsin enzyme activity in serum or the extracellular matrix often signifies a number of gross pathological conditions. Cathepsin-mediated diseases include: Alzheimer's, numerous types of cancer, autoimmune related diseases like arthritis and the accelerated breakdown of bone structure seen with osteoporosis. Up-regulated Cathepsin B and L activity has been linked to several types of cancer. These include cancer of the colon, pancreas, ovaries, breast, lung and skin (melanoma). Up-regulation of cathepsin K has been shown in lung tumors. Increased cathepsin K activity has also been linked to degenerative bone diseases including osteoporosis and post-menopausal osteoporosis.

Changes in concentration and activity of cathepsin B are observed in aging and under various pathophysiological conditions including rheumatoid arthritis , osteoarthritis , tumors and metastasis . Cathepsin B also stimulates angiogenesis in cancer tissues and degrades and inactivates the inhibitors of metalloproteases . Furthermore, up-regulated extracellular activity and relocalization of cathepsin B to the cell surface and into the extracellular matrix was observed in malignant tissues, suggesting its role in tumor invasion . Cathepsin B and its zymogen form are secreted, together with trypsinogen and active trypsin, into the pancreatic juice of patients with sporadic pancreatitis or hereditary pancreatitis.

In addition to being an endopeptidase, shows peptidyl-dipeptidase activity, liberating C-terminal dipeptides cathepsin B with tumor invasion and metastasis. Cathepsin B expression is increased in many human cancers at the mRNA, protein and activity levels. In addition, cathepsin B is frequently overexpressed in premalignant lesions, an observation that associates this protease with local invasive stages of cancer. Increased expression of cathepsin B in primary cancers, and especially in preneoplastic lesions, suggests that this enzyme might have pro-apoptotic features. Expression of cathepsin B is regulated at many different levels, from gene amplification, use of alternative promoters, increased transcription and alternative splicing, to increased stability and translatability of transcripts. During the transition to malignancy, a change in the localization of Cathepsin B occurs, as demonstrated by the presence of cathepsin B-containing vesicles at the cell periphery and at the basal pole of polarized cells. Due to increased expression of Cathepsin B and changes in intracellular trafficking, increased secretion of procathepsin B from tumors is observed. Active cathepsin B is also secreted from tumors, a mechanism likely to be facilitated by lysosomal exocytosis or extracellular processing by surface activators. Cathepsin B is localized to caveolae on the tumor surface, where binding to the annexin II heterotetramer occurs. Activation of cathepsin B on the cell surface leads to the regulation of downstream proteolytic cascade(s).

Cathepsin B is thought to play a central role in intrapancreatic trypsinogen activation and the onset of pancreatitis. Cathepsin B is abundantly present in the secretory compartment of the healthy human pancreas and is secreted together with trypsinogen and active trypsin into the pancreatic juice of patients with chronic pancreatitis. Cathepsin B mediates trypsinogen activation in pancreatitis. Once trypsin is activated, it can catalyze the activation of other digestive proenzymes as well as trypsinogen itself, initiating the autodigestion of the gland.

Cathepsin B contributes to cartilage destruction in osteoarthritis and pathological proteolysis in rheumatoid arthritis and cancer. Cathepsin B activity levels increase in the diseased state and may be an important target for designing small molecule inhibitors to reduce the inflammation and tissue destruction associated with rheumatoid arthritis.

Cathepsin B activity appears to play a critical role in lysosomal permeabilization in cytotoxic events, causing release of lysosomal proteases into the cytosol, which in turn cause mitochondrial dysfunction. The current findings extend these observations by demonstrating a role for this protease in vivo during a model of human liver disease, namely, cholestasis.

Arachidonic acid (AA) generated by cytosolic phospholipase A2 (cPLA2) has been suggested to function as a second messenger in tumor necrosis factor (TNF)-induced death signaling. Here, we show that cathepsin B-like proteases are required for the TNF-induced AA release in transformed cells. Pharmaceutical inhibitors of cathepsin B blocked TNF-induced AA release in human breast (MCF-7S1) and cervix (ME-180as) carcinoma as well as murine fibrosarcoma (WEHI-S) cells.

Current status of Cathepsin B

The physiological role and the pathological implications of the newly discovered cathepsins with the exception of cathepsin K are not fully understood. Due to the similarity of their tertiary structures, selectivity between the cathepsins is not very different. Furthermore, cathepsins B, H, L, F, C, X and O are ubiquitous such that they have a broad tissue distribution, but they may be involved in more specialized confirmed to result in a reduction in the severity of joint inflammation, and a reduction in joint destruction in adjuvant-induced arthritis in rats. In addition, the first evidence that rheumatoid arthritis synovial fluid contained extremely high activities of mature cathepsin B has recently been disclosed. Cathepsin B has also been implicated in the pathology of a number of other important human diseases, including cancer and neurodegenerative disorders.

CA074, is a specific and potent cathepsin B inhibitor. It is used in probes where mature cathepsin B activities are thought to be involved. But, it was recently reported that CA074 indiscriminately inhibited both cathepsins B and Cathepsin X. However, Menard et al . showed that CA074 was found to inactivate cathepsin B at least 34000-fold more efficiently than cathepsin X. Hence, it is demonstrated that specific inhibitors of cathepsin B or cathepsin X can be designed, taking advantage of the presence of the occluding loop in cathepsin B.

Two types of Cathepsin B inhibitors have been known from terrestrial microorganisms.

• Peptidyl aldehydes
• Epoxysuccinyl peptides

These inhibitors are generally non selective, since the members of the papain superfamily possess similar tertiary structures and have similar specificities in the hydrolysis of substrates.

Leupeptin

Leupeptin (antibiotic) represents a number of peptidyl aldehydes isolated from Streptomyces species, which is believed to inhibit the enzyme by forming a tetrahedral hemithioacetate between its aldehyde and the thiolate of the enzyme active site. However these are not selective inhibitors. It is a reversible inhibitor of serine and cysteine proteases e.g. plasmin, trypsin, papain, cathepsin B, thrombin, calcium-dependent protease calpain. The half maximal inhibitory concentration (ID ₅₀ ) depends on the protease and ranges from 0.5 to 10 µg/ml.

Leupeptin is a potent inhibitor of cathepsin B in vitro and is presumed to act in a similar manner in vivo. It is currently being used in several laboratories to examine the role of lysosomal proteinases (cathepsin B) in mouse models of muscular dystrophy. Leupeptin in adequate concentrations in vivo is a potent stimulator of Cathepsin B activity in striated muscle, heart, liver and kidney of the mouse. This paradoxical effect indicates that care is required in the interpretation of the results of the use of leupeptin as a Cathepsin B inhibitor in vivo and that its use as an antiprotease for therapeutic purposes may be limited. Studies on CBZ-Phe-Ala-CHN2 demonstrated that this agent, when administered in vivo, inhibited Cathepsin B in the tissues assayed.

Tokaramide A

Tokaramide A belongs to peptidyl aldehyde class. Tokaramide A , was isolated from the marine sponge Theonella aff. Mirabilis .

E-64

E-64 was reported from Aspergillus japonicus. In this case, trans- L-(SS)epoxysuccinic acid is the reactive group essential for inhibition. E-64 was first reported by Hanada et al . in 1978 and it is the most well known of this class of natural compounds, the epoxysuccinyl peptides.

Loxistatin

A derivative of E-64, loxistatin was tested in Japan for the treatment of muscular dystrophy, but development was stopped in 1992 in phase III clinical trial. Loxistatin was found to cause hepatic injury in rats. Virtually all the natural epoxysuccinyl peptide derivatives that have been isolated and reported possess a trans -dicarboxylic acid structural feature. The P2 position is usually occupied by either hydrophobic leucine (Leu) or isoleucine (Ile) residues, or by aromatic phenylalanine (Phe) or tyrosine (Tyr) residues. The P3 positions are usually C3, C4 or C5 linear CH2 spacers terminated with a basic functionality.

The epoxysuccinyl peptide derivatives showed little or no inhibitory activities against serine, metallo and aspartic proteases. Several of theses natural compounds have low toxicity and they do not possess any antimicrobial activity against bacteria and fungi. Calpains were weakly inhibited, whereas cathepsin B, cathepsin L and papain were strongly and irreversibly inhibited. Generally, the substituents at the P2 delineate their importance for the selectivity between cathepsin B and cathepsin L. Substrates with Leu or Ile substitution at the P2 position showed a preference for cathepsin B. (IC ₅₀ s in the range of 8-130 nM) over cathepsin L by about 5 to 8 times. But, cathepsin L (IC ₅₀ s in the range of 1 to 13 nM or ng/mL) was better inhibited than cathepsin B by 10 to 70 times, when the P2 substituent was Phe or Tyr.

In vivo effects of compounds a , b , and c were evaluated in a low-calcium-diet-fed mouse model. The maintenance of blood calcium level in calcium deficient mice has been regarded as a result of bone resorption. Thus in this model, inhibitors of bone resorption are expected to decrease the plasma calcium level. Subcutaneous injections of compounds a and b resulted in strong bone resorption inhibitory effects at a dose of 10 mg/kg as the plasma calcium levels were lowered to 60 and 68%, respectively, of the initial levels in 6 hours.

The synthetic epoxysuccinyl compound CA074 bind in the S’ subsites with the dipeptidyl moieties (Ile-Pro-OH) occupying the S1’ and S2’ subsites of cathepsin B. X-ray crystal structures of enzyme-substrate complexes clearly revealed that the 2 oxygens of the proline carboxylate are hydrogen bonded to the 2 histidine residues (His ¹¹⁰ and His ¹¹¹ ) in the occluding loop. This unique feature thus explains the high selectivity of prime site inhibitors of cathepsin B over the other members of the papain family.

The configuration at the epoxide ring and that of the neighbouring amino acid moieties greatly influence the potency of the inhibitors. The natural epoxysuccinyl compound E-64 that bind in the S subsites of the enzyme prefer the (S, S) configuration over the (R, R) at the epoxide moiety in order to exert higher inhibitory. However, the reverse is observed for those inhibitors that bind to the S’ subsites. Thus, compound - EtO- t Eps-Leu- Pro-OH structurally very similar to CA030 -EtO- t Eps-Ile-Pro-OH, with an (S, S) configuration at the oxirane ring is 13-fold less active than its (R, R) isomer. In addition, the affinity ratio (CB/P) for cathepsin B (CB) over papain (P) increased from about 7 to over 10000, and the affinity ratio (CB/CL) for cathepsin B (CB) over cathepsin L (CL) increased from 260 to 21800, respectively, in favor of the (R, R) diastereoisomer. It was just natural in the next stage to design a bispeptidyl inhibitor that possessed the features of an E-64 type binding residues in the S subsites and those of a CA074 type in the S’ subsites. In this class of inhibitors, the configurational requirements for optimal interaction with both the S and the S’ subsites are nonadditive. Thus, (S, S)- MeO-Gly-Gly-Leu t Eps-Leu-Pro-OH is 13-fold more potent than its (R,R) diastereoisomer. Compound MeO-Gly-Gly-Leu t Eps-Leu-Pro-OH is also a selective inhibitor and its preferences for cathepsin B over papain (CB/P) and cathepsin L (CB/CL) are 103 and 1262 times, respectively. Nevertheless, this family of (S,S)- MeO-Gly-Gly-Leu t Eps-Leu-Pro-OH epoxysuccinyl derivatives comprises of the most potent cathepsin B inhibitors reported so far. The parent compound MeO-Gly-Gly-Leu t Eps-Leu-Pro-OH was attached with a (CH ₂ ) ₅ spacer at the S subsite end and then conjugated with mono-(6-deoxy-6-amino)-cyclodextrin. The inhibitory potency of this cyclodextrin derivative for cathepsin B is only reduced by a factor of 0.7, but its selectivity against cathepsin L has doubled relative to parent MeO-Gly-Gly-Leu t Eps-Leu-Pro-OH. The cyclodextrin derivative only blocks about 10-20% of cathepsin B activity in intact cells at 1000-fold higher concentrations than those required for a complete inhibition of cathepsin B in cell lysates. Both of these compounds are not cell- permeable (MCF- 7 breast cancer cells, HaCaT-cells or fibroblasts) at concentrations needed for full inhibition of lysosomal cathepsin B. The cyclodextrin derivative is fully water-soluble and is demonstrated to only target extracellular and/or membrane bound cathepsin B under these conditions. In addition, cyclodextrin derivative can accommodate the cytotoxic drug methotrexate in its cyclodextrin cavity thus forming an inclusion complex. In another approach, the parent compound MeO-Gly-Gly-Leu t Eps-Leu-Pro-OH was attached with a  (CH ₂ ) ₆ spacer, then converted by acylation to a rhodamine B derivative or to a biotin derivative. These derivatives are as equipotent to cathepsin B as the parent inhibitor MeO-Gly-Gly-Leu t Eps-Leu-Pro-OH, but their respective selectivity against cathepsin L is almost 4-5 times higher. They are also virtually non cell-permeant. In addition, affinity blot analysis with the bionylated derivative allowed a highly sensitive, selective and non-radioactive detection of active cathepsin B. This class of inhibitors offers several advantages:

(a) They target only extracellular and/or membrane bound cathepsin B thus leaving the intracellular enzyme perform its housekeeping function,

(b) They can act as a site- directed drug carrier system where extracellular active cathepsin B is known to be present ( i.e ., certain cancers) by forming inclusion complexes with certain cytotoxic drugs, and

(c) They can be used as selective non-radioactive detection of active cathepsin B, thus          developing tool kits for the detection of cathepsin B activities in rheumatoid arthritis, and tumor invasion or metastasis are possibilities.

Yamamoto et al . have proposed a rule regarding compound (d) the atomic position of the oxirane carbon which participates in the covalent bond formation with the cysteine thiol, i.e ., the nucleophilic attack of cysteine thiolate will occur at the oxirane moiety that possesses the P’ substituent. The oxygen of the carbonyl group adjacent to the epoxide moiety of the P’ substituent sits in the oxyanion hole of the enzyme, which is formed by the side-chain amide of Gln23 and the backbone NH of Cys29. After the binding of the oxygen atom at the oxyanion hole, the Cys29 thiolate attacks the epoxide carbon atom which bears the P’ substituent.

To date, very few natural aziridinesuccinyl derivatives are known. Miraziridine A is an aziridine-2,3-dicarboxylic acid derivative isolated from the marine sponge Theonella aff. mirabilis . This compound is a rare natural product that inhibited cathepsin B with an IC50 value of 1.4 ug/mL. The inhibition of cysteine proteases by peptides containing aziridine-2, 3-dicarboxylic acid or cyclopropenone building blocks has been described in the literature. Epithiosuccinyl peptidyl compounds have yet to be reported.

High resolution X-ray crystal structure of compound (e) and cathepsin B complex unambiguously showed the formation of a thioimidate complex from the reaction of the cysteine thiol of the enzyme and the nitrile moiety of the inhibitor. In addition, results from the predicted structural model in silico were in very good agreement with the experimental results obtained from X-ray crystallography. However, as a cathepsin B inhibitor, was only 2- to 8-fold more or less selective for cathepsin S or L.  To enhance potency and selectivity against other cysteine proteases, Greenspan et al . took advantage of the presence of the occluding loop in the S’ region of the enzyme for the design of new inhibitors. Thus, a carboxylate group tethered from the P1 position was oriented to make a salt-bridge interaction with the histidine residues (His ¹¹⁰ and His ¹¹¹ ) located in the S2’ subsite of the enzyme. Through structural modeling, selective, potent and reversible inhibitors of cathepsin B spanning the S3 and the S2’ binding sites of the enzyme were designed. The presence of the carboxylate group at the P2’ position of the cathepsin B inhibitors clearly plays an important role on the potency and selectivity against the other cathepsins. This approach can potentially be extended to the design of potent and reversible cathepsin B inhibitors with a peptidomimetic or non-peptidic skeleton. In addition, the inclusion of a tether deriving from the P1 position and spanning into the S’ binding region of the enzyme opens the door for the development of a wider variety of inhibitors possessing terminal electrophilic warheads.

The need for ongoing development of new drugs needs no emphasis in light of the current global situation of health and disease. Traditionally, the process of drug development has revolved around a screening approach, as nobody knows which compound or approach could serve as a drug or therapy. Such almost blind screening approach is very time-consuming and laborious.

The shortcoming of traditional drug discovery; as well as the allure of a more deterministic approach to combating disease has led to the concept of "Rational drug design". It will be very interesting to see if a peptidomimetic, a structure based drug design and/or analogue based drug design approach can be achieved to obtain therapeutically useful compounds, as this will open a window of opportunity for the treatment of disease conditions caused by active cathepsin B.

References

1. Greenspan, P.D., Clark, et al . Identification of Dipeptidyl Nitriles as Potent and Selective Inhibitors of Cathepsin B Through Structure-Based Drug Design J. Med. Chem., v44 pp.4524 , 2001
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About Authors:

Mr. L. Jayashankar ^a,* , Shruti Awasthi ^b , Madhulika Ganguri ^b , Prof. B. Syama Sundar ^c

^{* a} Research Scholar(Pharmaceutical chemistry), Department of Chemistry, Acharya Nagarjuna University , Guntur .

^b Research Associate, GVK Bio Sciences, Informatics Division, Hyderabad

^c Professor, Department of Chemistry, Acharya Nagarjuna University , Guntur

Mr. L. Jayashankar

*For Correspondence: L. Jayashankar, c/o. Prof. B. Syama Sundar, Department of Chemistry, Acharya Nagarjuna University , Guntur . E-mail: l.jayashankar@gmail.com, Phone: 9810043362

Shruti Awasthi

Madhulika Ganguri

B. Syama Sundar