SUMMARY AND CONCLUSION

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2 Leishmania parasites are a major public health risk in many countries and cause a wide spectrum of clinical disease referred to as leishmaniasis. The three major forms of diseases caused by them include cutaneous leishmaniasis, mucocutaneous leishmaniasis and fatal visceral infections. An estimated population of 15 million people is infected, with a further 350 million at risk in tropical and sub-tropical regions of the world. Ninety percent of all visceral leishmaniasis cases occur in Bangladesh, Brazil, India and Sudan. Worldwide distribution of the parasite greatly limits the means to control its spread. No effective vaccines are available against Leishmania infection as yet and treatment lies in chemotherapy. Pentavalent antimonials (First line drug), Amphotericine-B and Pentamidine (Second line drug) are effective drugs for leishmaniasis. Unfortunately, the increasing prevalence of parasites resistance to this first line drug, and the cost and the toxicity of the second line ones, have deteriorated the situation and warrants the development of new chemotherapeutic agents. Current hope for new chemotherapeutic and vaccination therapies lies in research program oriented towards identification of key process essential for parasite growth, survival in mammalian host and transmission by its insect vector. The Leishmania life cycle consists of two morphologically distinct stages: intracellular amastigotes that reside in the phagolysosome of mammalian macrophages, and extracellular promastigotes that reside within the gut of the sandtly vector. It is the amastigote stage that is able to withstand the hostile environment of macrophages and is responsible for pathogenesis. It is proposed that parasite expresses a range of developmentally regulated genes presumably with a critical role in parasite survival in that particular environment. Identification of those genes could help to identify new targets for controlling the diseases caused by this human pathogen. DNA microarray is a powerful method to study global gene expression in terms of quantitation of mrna levels. In the present study Leishmania genomic microarray containing 2304 PCR amplified DNA inserts were hybridized with tluorescent dye Cy-3 and Cy-5 labeled c-dna

3 generated from total RNA isolated from promastigote and axenic amastigotes respectively with an aim to identify differentially expressed genes. Analysis of the hybridization led to the identification of 21 genes, which exhibited differential expression in the two stages of parasite. However, none of them exhibited more than 2 fold up or down regulation in amatigote stage of parasite. Among 12 contigs, which showed upregulation in amastigote stage, BLAST analysis identified three clones 15D9, 10B5 and 38H5 exhibiting strong homology with interesting proteins namely Dipepidyl Carboxypeptidase, sodium stibogluconate resistant protein and Cytochrome p-450, respectively. These genes have not been studied in Leishmania so far. Therefore, sodium stibogluconate resistant protein gene and dipepidyl carboxypeptidase gene were selected for detailed characterization The complete open reading frame (or}) of sodium stibogluconate resistant protein of L. donovani (LdSSRP) of 1866 bp was PCR amplified using genomic DNA isolated from log phase promastigotes (Dd8 starain) and primers, designed on the basis of available L. major genome database ( The product was cloned in pcr-topon vector and sequence was determined by primer walking. LdSSRP coding region was found enriched in G+C residues (61.3%) in comparison with A+T residues (38.7%) like other Leishmania genes. It exhibited strong sequence identity with L.major sodium stibogluconate resistant protein (LmjF ), which is present at chromosome no.31. The gene encodes for 621 amino acids with predicted molecular weight of Iilla. The average hydropathy of L. donovani SSG (LdSSG) is suggestive of a hydrophilic protein. No signal peptide sequence or trans-membrane domains were observed in putative LdSSG. The gene product of LdSSRP or! did not show significant homology to other known proteins in the data banks but gene was present among different Leishmania species like L.major and L.terentole. Unfortunately, this lack of homology does not provide insights on the functionality of protein. The gene was expressed and purified to homogeneity under denaturating conditions as more than 90% of recombinant protein was expressed in inclusion bodies, even at low temperature (22 C). Thus a gene which was earlier shown to be amplified in antimony resistant mutants of L. tarentolae, was for the first time identified as differentially expressed gene in amastigote stage of parasite. The gene has been cloned, expressed and the recombinant protein was 114

4 purified to be used in future studies to explore its role in pathogenesis or resistance, if any. Another selected gene for detailed characterization was dipeptidylcarhoxypeptidase. Todate, it has been reported, cloned and characterized only in bacteria. It cleaves dipeptides off the C-termini of various peptides and proteins, the smallest substrate being N-blocked tripeptides and unblocked tetra peptides. In the mammalian system, the same function is performed by peptidyl dipeptidase A, also known as angiotensin converting enzyme (ACE), which acts on many substrates, primarily releases a C-terminal dipeptide from angiotensin I to produce a potent vasopressor, angiotensin II. In the present study, we have identified for the first time in any protozoan parasite a gene encoding dipeptidylcarboxypeptidase that showed -2-fold up-regulation in microarray analysis in axenic amastigotes. Full length coding sequence of the DCP gene was amplified by PCR from L. donovani genomic DNA using primers designed from L. major DCP sequence annotated in GenBank. Primary structure analysis of an open reading frame of LdDCP of 2037 bp revealed the presence of an active Zn binding site, which is a characteristic of Zn +2 metallopeptidases. The nucleotide sequence of LdDCP has been deposited in GenBank under Accession No. AAV LdDCP coding region was found enriched in G+C residues (61.4%) in comparison with A+T residues (38.6%). LdDCP shows strong sequence identity with L. major dipeptidylcarboxypeptidase (LmjF ), which is present at chromosome no.2. The ORF encodes for a polypeptide of 678 amino acids with predicted molecular mass of 76,547Da and predicted pi of The average hydropathy of L. donovani DCP (LdDCP) is suggestive of a hydrophilic protein. No signal peptide sequence or trans-membrane domains were observed in putative LdDCP. The calculated percent similarity shows that LdDCP has >92% homology to L. major putative DCP, % homology to bacterial peptidyl dipeptidase, 45% to bacterial DCP and 29-32% to bacterial oligopeptidase A (OpD A). The N-terminal domain is much less conserved compared to the C-terminal domain, with all three conserved motifs. In phylogenetic analysis, mammalian metallopeptidases and bacterial oligopeptidases are grouped as independent clusters. Whereas, all the dipeptidylcarboxypeptidase including L. donovani 115

5 DCP and peptidyl dipeptidase are grouped in to a separate cluster indicating similarity between the genes. The Southern blot analysis with a C-terminal LdDCP probe (that contains conserved amino acid motifs including the Zn binding site) suggests that there is more than one DCP/DCP-related gene in the L. donovani genome. Expression of the LdDCP gene as determined by northern blot analysis confirmed that LdDCP was up regulated in axenic amastigotes compared to promastigotes.the presence of two bands in northern analysis further supports the suggestion of a second gene with homology to sequences in LdDCP. In order to assess the expression in Leishmania cells, polyclonal serum was raised against a recombinant LdDCP protein expressed in E. coli (rlddcp). The rlddcp was expressed as a histidine tagged protein and purified under native conditions from E. coli lysates by nickel agarose affinity chromatography. Analysis of the purified rlddcp fraction by SDS-PAGE and Coomassie blue staining showed that the major protein corresponds to the molecular weight predicted by the open reading frame plus the size of the epitope tag and six histidines added in the recombinant LdDCP construct. This preparation was used to generate a rabbit polyclonal a-lddcp antibody. Western blot analysis showed that, the a-lddcp antibody reacted with a single ~ 77kDa protein in lysates of L. donovani promastigotes and axenic amastigotes. These results showed that LdDCP was expressed by the two stages of the parasite. However, the intensity of the DCP band was ~2-fold higher in axenic amastigotes compared to promastigotes. The protein levels on western blot are in agreement with micro~rray and northern blot results described above and indicate that DCP is probably expressed more in the amastigote stage of the parasite. To determine whether the putative LdDCP gene codes for an active DCP enzyme, the purified rlddcp was assayed for DCP enzymatic activities. The recombinant enzyme showed carboxy-dipeptidase activity with synthetic substrates for ACE i.e Hip-His-Leu (HHL). LdDCP has been found highly unstable at room temperature or at 4 C, therefore, stability studies were performed in presence of stabilizing agent (Glycerol, Tween-20 & BSA). 50% glycerol at -20 C was found as most suitable storage conditions. DCP followed Michaelis-Menten kinetics for the substrate HHL. Kinetic parameters Km and Vmax values of LdDCP were 4mM and J..lmole/mllmin respectively. The L. 116

6 donovani DCP had a wide range of ph and retained its maximum activity in the range of LdDCP is a metal dependent enzyme and activity can be inhibited in presence of Ni+ 2, Cu+2 and Zn+2 or stimulated in presence of Ca+2, Mn+2, Mg+2 and Co+2 ions. The enzyme was more sensitive to chelator 1,10 phenathroline than EDT A. Further, results indicated that E64 was a potent inhibitor for LdDCP as compared to trypsin inhibitor. Circular dichroism spectra analysis revealed that LdDCP is predominantly a- helix (61 %) in nature. DCP of L. donovani closely resemble mammalian ACE in there substrate specificity and their susceptibility to chemical inhibitors like captopril and pglu-trp-pro-arg-pro-gln IIe-Pro-Pro (bradykinin potentiating peptide, BBPI). Analysis of Dixon plot revealed a competitive mode of inhibition, and reversible inhibition constant (Ki) was determined to be 35.8nM for captopril. Further, hydrolysis of HHL by LdDCP was also inhibited by BPPI in competitive manner with a Ki value of 3.9~M. However, these values were several folds higher than value reported for human testicular ACE. It clearly demonstrates that the captopril inhibits LdDCP and ACE in different magnitude although both peptidases belong to same group (M3 family of mono zinc peptidase). To determine whether blocking DCP activity could be used as a means to control parasite growth, captopril in various concentrations was added to Leishmania promastigotes in culture. The drug exhibited dose dependent inhibition of promastigote growth with 1Cso ~g/ml. Thus, biological activity of the drug is correlated well with its ability to inhibit parasite DCP. The data strongly suggested this newly identified DCP could serve as drug target in Leishmania. To rationalize observed differences in experimentally determined values for dipeptidycarboxypeptidases from L. donovani and Human, it is essential to know the binding property of substrate or inhibitor to the enzyme. It might be possible that though, LdDCP, EcDCP and ACE, share high degree of conservation in critical active site but there may be considerable sequence variation in neighbouring active site residues which is responsible for structural differences in shape and conformation of their active sites leading to catalytic variation. These differences can be exploited in rational drug designing. Therefore, a three dimensional model structure of LdDCP was constructed using EcDCP as template, by means of comparative modeling through multiple sequence 117

7 alignment followed by intensive optimization and validation. The resulting model demonstrated a reasonable topology as judged from Ramachandran plot and acceptable three diamentional structure compatibility as assessed by the profiles-3d score. The modeled monomer exhibited two separate sub domains with an active site at the interface It is predominantly 57% a-helical and 6% ~-secondary in nature, which is comparable of secondary structure determined by circular dichroism spectroscopic measurements. Captopril, a known ACE inhibitor was docked into active sites of ACE, EcDCP and LdDCP. The docked scores correlated reasonably well with experimental values. The molecular electrostatic potentials (MEP) further suggested potentially important structural differences between the active sites of the three functionally similar enzymes. Although LdDCP and EcDCP appear to have a similar shape, but the cavity groove of LdDCP is more electronegative compared to EcDCP. Further, the shape and electrostatic potential covering the active site residues in ACE was entirely different (mostly electropositive residues are predominant in active site) as compared to LdDCP or EcDCP. These differences may account for differential recognition and binding pattern of captopril with ACE, EcDCP and LdDCP. Results of current study will be useful in the early design and development of inhibitors by either de novo drug design or virtual screening of large small molecule databases leading to development of new antileishmanial agents. Virtual screening was performed in an attempt to identify novel drug like non-peptide inhibitors of parasitic dipeptidylcarboxypeptidase. The CDRI repository has pure compounds collected over four decades from in-house chemical synthesis. All compounds were screened against homology models of dipeptidylcarboxypeptidase in two consecutive stages of docking. A total of 46 diverse inhibitors were identified from an initial group of 103, of which 6 compounds appeared to be inhibitors of dipeptidylcarboxypeptidase. All six compounds inhibit promastigote growth in a dose dependent manner. To date dipeptidylcarboxypeptidase has been characterized only in bacteria where, a major fraction of DCP is conti ned to the cytoplasm. Being an intracellular enzyme, the major function assigned in bacteria for DCP is in catabolism i.e. in the degradation of intracellular proteins. The same function can be proposed for leishmanial DCP. In 118

8 Leishmania, differentiation of promastigote to amastigote is accompanied by a tremendous increase in total protease activity which is sustained even after complete differentiation. These proteases/peptidases act on large proteins to release small peptides that can be further degraded by DCP. The upregulation of DCP in the amastigote stage is therefore in accordance with an increase in total protease activity. Due to broader substrate specificity than aminopeptidase or oligopeptidase B, LdDCP may help in processing of small peptides released by a variety of endopeptidases during the life cycle of the parasite. Thus, LdDCP may have a role in parasite stage differentiation and is expected to play an indirect role in nutrition and pathogenesis. Taken together, the presence of LdDCP in all life cycle stages of the parasite, its emergence into a unique subgroup of M3 metallopeptidases, differential inhibition by ACE inhibitors and parasite growth by enzyme inhibitor captopril, established that this newly identified DCP could serve as drug target in Leishmania. Further, newly identified molecules which selectively inhibits LdDCP and also parasite growth has opened new vistas/ leads for optimization into more potent and efficacious drug candidates to treat these protozoal infections. 119