Curative efficacy of purified serine protease inhibitor PTF3 from potato tuber in experimental visceral leishmaniasis.

To overcome the drug toxicity and frequent resistance of parasites against the conventional drugs for the healing of human visceral leishmaniasis, innovative plant derived antileishmanial components are very imperative. Fuelled by the complications of clinically available antileishmanial drugs, a novel potato serine protease inhibitor was identified with its efficacy on experimental visceral leishmaniasis (VL). The serine protease inhibitors from potato tuber extract (PTEx) bearing molecular mass of 39 kDa (PTF1), 23 kDa (PTF2) and 17 kDa (PTF3) were purified and identified. Among them, PTF3 was selected as the most active inhibitor (IC50 143.5 ± 2.4 µg/ml) regarding its antileishmanial property. Again, intracellular amastigote load was reduced upto 83.1 ± 1.7% in pre-treated parasite and 88.5 ± 0.5% in in vivo model with effective dose of PTF3. Protective immune response by PTF3 was noted with increased production of antimicrobial substances and up-regulation of pro-inflammatory cytokines. Therapeutic potency of PTF3 is also followed by 80% survival in infected hamster. The peptide mass fingerprint (MALDI-TOF) results showed similarity of PTF3 with serine protease inhibitors database. Altogether, these results strongly propose the effectiveness of PTF3 as potent immunomodulatory therapeutics for controlling VL.

White jute (Corchorus capsularis L.) leaf extract has potent leishmanicidal activity against Leishmania donovani.

In pursuit of effective, safe and affordable antileishmanial drugs, the current study was designed to explore Corchorus capsularis L. leaf extract (CCEx) as an effective leishmanicidal substitute against Leishmania donovani. The leaf extract displays potent antileishmanial activity against L. donovani promastigotes with an IC50 value of 79.00 ±â€¯0.3 µg/ml. CCEx also significantly induces intracellular reactive oxygen species (ROS) with a concomitant decrease in the level of non-protein thiols in virulent parasites. Additionally, CCEx treatment induces substantial morphological alterations in parasites. Moreover, reagent-based phytochemical analysis of the extract revealed the presence of various phytochemical constituents. Further study is underway to identify the bioactive component(s) or fraction(s) of CCEx through bioassay-guided fractionation.

Serine protease inhibitors rich Coccinia grandis (L.) Voigt leaf extract induces protective immune responses in murine visceral leishmaniasis.

Leishmaniasis is a parasite-mediated tropical disease affecting millions of individuals worldwide. The available antileishmanial chemotherapeutic modalities exhibit adverse toxicity, exorbitant price and advent of drug-resistant parasites. Hence, plant-derived products are an alternative preference for the emergence of novel and effective antileishmanial agents that rejuvenate the host immunity with limited toxicity. The present work is complementary to our previous report that revealed the in vitro antileishmanial and immunomodulatory activity of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) rich in serine protease inhibitors. Thus, preliminary objectives of the study were to elucidate the leishmanicidal activity and host effector mechanism in Leishmania donovani infected BALB/c mice treated with Cg-Ex. Oral administration of Cg-Ex significantly reduced the spleen and liver parasite burden at dose-dependently. The parasite elimination was associated with generation of ROS and NO that are interrelated with up-regulation of disease-suppressing Th1 cytokines and down-regulation of disease-promoting Th2 cytokines at both protein and mRNA level. Moreover, Cg-Ex augmented the delayed-type hypersensitivity (DTH) response and serum IgG2a level which are correlated with the diminution of parasite burden with no hepatic and renal toxicity. Additionally, histological analysis of spleen depicted the improvement of structural disorganization of white and red pulp after Cg-Ex treatment. Therefore, our intriguing findings have presented the first indication of in vivo antileishmanial efficacy through activation of pro-inflammatory immune responses of the host by a natural plant leaf extract (Cg-Ex) containing serine protease inhibitors which could have a role as a potential immunomodulator against visceral leishmaniasis.

Functional attribution of LdISP, an endogenous serine protease inhibitor from Leishmania donovani in promoting infection.

Leishmaniasis, a parasitic disease caused by unicellular eukaryotic protozoa of the genus Leishmania, affects more than 12 million people worldwide. Events of leishmaniasis are based on the infection of the mammalian host, precisely macrophages, where both host and parasite derived proteases and endogenous inhibitors are significant. Pathogen derived protease inhibitors have generated considerable interest as they often act as an agent promoting infection and parasitic survivability. An endogenous serine protease inhibitor from Indian strain of Leishmania donovani was previously identified by our group and named as LdISP. It has been found to inhibit neutrophil elastase (NE), responsible for natural inflammation process. However, LdISP's role in progression of infection or the proteomics based structural exposition has not been explored. The present study is aimed to localize and validate the potential role of LdISP in infectivity. We found that LdISP localized endogenously and treatment of infected host cells with LdISP curbs ROS and NO production. Additionally, in silico studies are carried out to predict the putative amino acid residues of LdISP involved in the inhibition process. Taken together, our results demonstrate that LdISP eventually exerts a pronounced role in L. donovani infection.

Leishmania donovani serine protease encapsulated in liposome elicits protective immunity in experimental visceral leishmaniasis.

This study is aimed to evaluate the protective effect of L. donovani intracellular serine protease (SP-Ld) in combination with Freund's adjuvant and liposomal formulations against experimental visceral leishmaniasis (VL). The animals were immunized with SP-Ld in combination with adjuvant and evaluated for its immunogenicity and protective efficacy against Leishmania donovani. The infection was initially assessed by microscopic examination. Immunogenicity of SP-Ld was measured by detecting protease specific-IgG, IgG1 and IgG2a levels by ELISA. Cytokines levels were measured by ELISA and Reverse Transcription Polymerase Chain Reaction (RT-PCR). The vaccine efficacy of SP-Ld was also evaluated by measuring antibody response and survival potency in hamster model. SP-Ld vaccinated Balb/c mice resulted significant reduction of parasite burden with increased levels of IgG2a and decreased levels of IgG1. SP-Ld vaccination also induced Th1 type immune response with the rise of IL-12, IFN-γ and TNF-α with decreased levels of IL-10 and TGF-ß. Importantly, liposomal incorporated SP-Ld exerted better protection rather than in combination with Freund's adjuvant. Additionally, liposome encapsulated SP-Ld vaccinated hamsters continued to survive beyond 8 months against virulent L. donovani post challenge. Overall, these findings demonstrated SP-Ld as an effective immunogen which opens a new perspective for the generation of potential vaccine candidate against leishmaniasis.

Coccinia grandis (L.) Voigt Leaf Extract Exhibits Antileishmanial Effect Through Pro-inflammatory Response: An In Vitro Study.

The conventional drugs used for the treatment of human visceral leishmaniasis have concerns about the toxicity and most importantly parasite resistance. To overcome these troubles, more efforts are made for the development of innovative therapeutic agents having effective antileishmanial activity and simultaneously stimulate adaptive immune system of host cells. Hence, search for new leishmanicidal from the natural origin like plants has shown its effectiveness for the treatment of this tropical disease. The aim of this study is to investigate and characterize the antileishmanial efficacy of Coccinia grandis (L.) Voigt leaf extract (Cg-Ex) with its immunomodulatory property against Leishmania donovani in an in vitro experimental model. Cg-Ex significantly reduces the intracellular L. donovani parasite load with IC50 value 193 ± 0.78 µg/ml, but it has lower cytotoxicity on the murine RAW 264.7 macrophage cell line. Interestingly, Cg-Ex induces the generation of potent antimicrobials like reactive oxygen species and nitric oxide dose dependently in infected murine macrophages. Moreover, the increased production of Th1 cytokines (IL-12, TNF-α) with a concurrent decrease of Th2 cytokines (IL-10, TGF-ß) was also observed in Cg-Ex-treated infected host macrophages. Our results thus confirm that serine protease inhibitor(s)-rich Cg-Ex exhibits antileishmanial activity in vitro, and this was mediated through the modulation of pro-inflammatory cytokines. On the whole, the present findings first demonstrate the antileishmanial property of Cg-Ex targeting the Leishmania serine protease resulting protection of host cells with Th1 cytokine expression. Thus, these data indicate that C. grandis leaf extract (Cg-Ex) might be considered as a new lead for designing alternative and novel natural therapeutic against visceral leishmaniasis.

Protective inflammatory response against visceral leishmaniasis with potato tuber extract: A new approach of successful therapy.

The increasing number of drug resistance issue of Leishmania donovani strain to common drugs compels to develop new therapeutics against leishmaniasis with minimal toxicity. In this regard, bioactive phytocomponents may lead to the discovery of new medicines with appropriate efficiency. The important roles of Leishmania proteases in the virulence of Leishmania parasite make them very hopeful targets for the improvement of current remedial of leishmaniasis. As part of a hunt for new drugs, we have evaluated in vivo anti-leishmanial activity of serine protease inhibitor rich fraction (PTEx), isolated by sodium bisulfite extraction from potato tuber. The amastigote load of 25mg/kg body weight/day treated BALB/c mice showed 86.9% decrease in liver and 88.7% in case of spleen. This anti-leishmanial effect was also supported by PTEx induced immunomodulatory activity like acute formation of ROS and prolonged NO generation. The Th1/Th2 cytokine balance in splenocytes of PTEx treated animals was estimated and evaluated by ELISA assay as well as by mRNA expression using RT-PCR. Furthermore, significant survival rate (80%) was observed in PTEx treated hamsters. Thus, from the present observations we could accentuate the potential of PTEx to be employed as a new therapeutics from natural source against L. donovani. This might also provide a novel perception of natural serine protease inhibitor from potato tuber as an alternate approach for the treatment of visceral leishmaniasis.

Antiproteolytic and leishmanicidal activity of Coccinia grandis (L.) Voigt leaf extract against Leishmania donovani promastigotes.

In visceral leishmaniasis (VL), development of alternative safe therapeutic strategy is gaining paramount wherein natural components of plant origin have prominence. We explored Coccinia grandis (L.) Voigt, a medicinal plant known in traditional folk medicine, for its antileishmanial efficacy. SDS-PAGE analysis of the C. grandis leaf extract (Cg-Ex) showed few protein bands about 14-66 kDa among which three (64.8, 55.8 and 15.3 kDa) were identified as serine protease inhibitors by reverse zymography. Since the virulence of Leishmania is also attributed by serine proteases, objective of the present study was to evaluate in vitro antileishmanial activity of Cg-Ex, targeting Leishmania donovani serine protease(s). Inhibition study of Cg-Ex in gelatin-zymogram and spectrophotometric assay revealed its strong inhibitory activity against bovine trypsin rather than chymotrypsin, and also showed significant inhibition of L. donovani serine protease(s). Further, studies with Cg-Ex were extended to estimate its antileishmanial efficacy with half maximal inhibitory concentration (IC50) at 308.0 ± 2.42 µg/ml along with significant morphological alterations. The results have demonstrated the potential of the serine protease inhibitor rich fraction of the C. grandis leaf extract against visceral leishmaniasis.

In vitro anti-leishmanial efficacy of potato tuber extract (PTEx): leishmanial serine protease(s) as putative target.

Leishmaniasis, a neglected tropical disease (NTD) causes major health problems in the tropical and subtropical world. Most of the antileishmanial modern therapies with different formulations of pentavalent antimonials, Miltefosine, Amphotericin B etc. are not satisfactory in recent times due to high toxicity to the host and present rising strain resistance issues. So there is an urgent need to develop new, safe and cost-effective drugs against leishmaniasis. In this regard, bioactive phytocomponents may lead to the discovery of new medicines with appropriate efficiency. The prominent roles played by Leishmania proteases in the virulence of this parasite make them very promising targets for the development of current therapeutics of leishmaniasis. As part of a search for novel drugs, we have evaluated in vitro anti-leishmanial activity of serine protease inhibitor rich fraction (PTEx) obtained from potato tuber. The extract (PTEx) was prepared by sodium bisulfite fractionation and inhibitors were identified by reverse zymography. Inhibition study of PTEx in gelatin-zymogram and spectrophotometric assay using BApNA and BTpNA as substrate reveal its strong inhibitory activity against trypsin as well as serine proteases present in cell lysate of Leishmania donovani infective strain. The in vitro MTT based colorimetric assay as well as ex vivo L. donovani infected macrophages showed reduced parasite viability and intracellular parasite load with IC50 = 312.5 ± 0.1 µg/ml and IC50 82.3 ± 0.2 µg/ml of PTEx respectively in a concentration dependent manner. This anti-leishmanial effect was also preceded by PTEx induced acute formation of ROS and prolonged NO generation. The PTEx has no significant cytotoxic effect on host macrophages. So taken together, these findings indicate that PTEx has promising leishmanicidal effect and thus this study provides a new perspective of natural serine protease inhibitor from potato tuber on the development of new drug against leishmaniasis.

Protease inhibitors in potential drug development for Leishmaniasis.

Leishmaniasis is a deadly protozoan parasitic disease affecting millions of people worldwide. The treatment strategy of Leishmania infection depends exclusively on chemotherapy till date. But the treatment of the disease is greatly hampered due to high cost, toxicity of the available drugs and more importantly emergence of drug resistance. Hence the potential new drugs are highly needed to combat this disease. The first and foremost step of the drug discovery process is to search and select the putative target in a specific biological pathway in the parasite that should be either unambiguously absent in the host or considerably different from the host homolog. Importantly, Leishmania genome sequences enrich our knowledge about Leishmania and simultaneously reinforce us to identify the ideal drug targets that distinctly exist in the parasite as well as to develop the effective drugs for leishmaniasis. Though the leishmanial research has significantly progressed during the past two decades, the identification of suitable drug targets or development of effective drugs to combat leishmaniasis is far from satisfactory. Enzymatic systems of Leishmania metabolic and biochemical pathways are essential for their survival and infection. Concurrently, it is noteworthy that Leishmania proteases, especially the cysteine proteases, metalloproteases and serine proteases have been extensively investigated and found to be indispensable for the survival of the parasites and disease pathogenesis. Herein, we have discussed the importance of few enzymes, particularly the Leishmania proteases and their inhibitors as promising candidates for potential development of anti-leishmanial drugs.

Implications of calpains in health and diseases.

The number of mammalian calpain protease family members has grown as many as 15 till recent count. Although initially described as a cytosolic protease, calpains have now been found in almost all subcellular locations i.e., from mitochondria to endoplasmic reticulum and from caveolae to Golgi bodies. Importantly, some calpains do not possess the 28 kDa regulatory subunit and have only the 80 kDa catalytic subunit. In some instances, the 80 kDa subunit by itself confers the calpain proteolytic activity. Calpains have been shown to be involved in a number of physiological processes such as cell cycle progression, remodeling of cytoskeletal-cell membrane attachments, signal transduction, gene expression and apoptosis. Recent studies have linked calpain deficiencies or it's over production with a variety of diseases, such as muscular dystrophies, gastropathy, diabetes, Alzheimer's and Parkinson's diseases, atherosclerosis and pulmonary hypertension. Herein, we present a brief overview on some implications of calpains on human health and some diseases.