Development of therapeutics targeted to DNA topoisomerases of Leishmania

Hemanta K. Majumder

Infectious Disease and ImmunologyIndian Institute of Chemical Biology

IACIBDHAKA, Bangladesh

Sept 1-2, 2012

Leishmania: a unicellular kinetoplastid protozoan parasite

The long slender and motile PROMASTIGOTES reside in the sandfly gut

The round, sessile AMASTIGOTES reside inside host macrophages

Leishmaniasis - Clinical Spectrum

Visceral Leishmaniasis (VL)

Post Kala-azar dermal Leishmaniasis (PKDL)

Cutaneous and Diffused cutaneous Leishmaniasis

(Cl, DCL) Mucocutaneous Leishmaniasis (MCL)

Leishmaniasis - Chemotherapy

L. donovani infection induces Immunosuppressive effect, no host response

No effective vaccine is available till date Chemotherapy, the ONLY choice

Urea Stibamine, Sodium StibogluconateIncreasing toxicity & serious side-effects.

Pentamidine, Amphotericin B Toxic, High cure rate, very expensive

Allopurinol, WR6026, Ketoconazole, Miltefosine Toxic, High doses of efficacy, Under clinical trial (phase IV)

Mechanism of Topoisomerase

DNA Topoisomerases / ‘Cells Magicians’

Ubiquitous enzymes, involved in interconversion of topological isomers of DNA molecules

XInvolved in vital cellular processes, e.g. replication, transcription, recombination, chromosomal segregation etc.

XMainly of two types; Type I & Type II.

Type I

Makes single stranded nicks, does not require ATP

Type II

Makes double stranded nicks, requires ATP

X Possesses an inherent dark side capable of inflicting great harm to the genome of an organism.

X Topoisomerase inhibitors have emerged as anti cancer, anti bacterial and anti parasitic agents.

Leishmania donovani bi-subunit topoisomerase I “A missing link”

in evolution of type IB enzyme

Biochem J. 2008; 409 : 611-22

Indole 3 Carbinol

3,3’ DiindolylmethaneStructure of DIM

DIM is a potent inhibitor ofLeishmania donovani

topoisomerase IB inhibitor

AcidCondensation

DIM : a Potent Inhibitor of LdTOP1LS

Mol Pharmacol. 2008 ; 74 : 1292-307

Microscopic analysis of in vitro cytotoxicity of DIM. A, log phase promastigote cells (3 x106) were cultured for 12 h in M199 media supplemented with 10% FBS and treated with 1, 5, 10, 15, and 20 M DIM for 2, 4, 8, and 12 h. Percentage of viable promastigotes was measured by MTT assay

L. major, L. tropica, and L. tarentolae promastigotes were also cultured in the presence of 15 M DIM for 2, 4, 8, and 12 h. The percentage of viable promastigotes was measured by MTT assay.

DIM inhibits Leishmania spp. growth

DIM Reduces the Parasites Burden in infected golden hamsters

Mol Pharmacol. 2008 ; 74 : 1292-307

A. externalization of phosphatidyl serine was detected in L. donovani promastigote cells. Annexin V labeling of L. donovani promastigotes was measured after treatment with 0.2% DMSO (1), with DIM (15 mM) for 4 and 6 h (2 and 3), and with CED3/CPP32 group of protease inhibitor (VAD-fmk) before reatment with DIM (4).

B. apoptotic cells detected by flow cytometric analysis using annexin V and PI in FL-1 versus FL-2 channels.

C. bar diagram showing the viable cells, apoptotic cells, and necrotic cells as determined by fluorescence-activated cell sorting analysis. representative data from one set of experiments are expressed as means ± S.D.

DIM induces Apoptotic events in Leishmania cells

The growth of parasites (WT and strain LdDR50) was monitored in the presence of increasing concentrations (1, 5, 10, 15, and 20 M) of DIM at 24 h. The level of proliferation of the WT Leishmania strain in the absence of any treatment was considered the maximal-growth control.

The accumulation of DIM was measured at given time intervals by spectrophotometric analysis at absorbance maxima of 280 nm as described in Materials and Methods. Data are means standard errors from three independent experiments

Effects of DIM on the growth of WT and LdDR50 parasites

Antimicrob Agents Chemother. 2009; 53 :2589-98

Resistance to DIM causes Random Mutations in Topoisomerase Gene

Close-up view of the active-site Tyr222 of L. donovani topoisomerase I from WT parasites based on the crystal structure of L. donovani topoisomerase I. The effect of the F270L mutation in the large subunit of L. donovani topoisomerase I was modeled using the Swiss-Pdb Viewer, version 3.7.

Antimicrob Agents Chemother. 2009; 53 :2589-98

Effects of DIM on the growth of

LdTOP1L and LdTOP1S mutant

parasites. The parasites were

grown in the presence of

increasing concentrations of

DIM (1 to 20 mM) for 24 h. WT

parasites were transfected with

mutated genes encoding

different mutations as indicated.

Effect of DIM on Different LdTOP1LS Mutant

Antimicrob Agents Chemother. 2009; 53 :2589-98

Effects of DIM on the relaxation activities of WT and site-specific mutated topoisomerase I enzymes. The sensitivities to DIM of WT and mutant topoisomerase I enzymes were determined by a plasmid DNA relaxation assay.

Effect of DIM on Different Site Directed Mutations of LdTOP1LS

Development of Derivatives of 3, 3'-Diindolylmethane as Potent Leishmania donovani Bi-Subunit Topoisomerase IB Poisons

Emergence of resistance against DIM for leishmaniasis poses great problem in killing the Leishmania cells

We have synthesized several derivatives of DIM to ensure the killing of these mutated strains

PLoS One. 2011;6(12):e28493

Effect of DIM-derivatives on Leishmania parasites

Growth of L. donovani AG83 promastigotes (A) and DIM resistant promastigotes (B) were monitored in presence of increasing concentrations (1, 5, 10, 20 and 50 mM) of DIM, DPDIM, DMDIM and DMODIM for 12 h.

Proliferation of Leishmania strain in absence of treatment was considered as a maximal growth control.

(C) Change in IC50 values after a prolong exposure to DIM derivatives has been assessed.After every 5 day a small aliquot was taken out and number of viable promastigotes was counted for each concentration. IC50 values have been determined from them using Graph Pad Prism ver 5.0 software and a graphical plot was generated over time.

PLoS One. 2011;6(12):e28493

PLoS One. 2011;6(12):e28493

Effectiveness of clearance of internalized L. donovani (AG83) from in vitro infected mouse peritoneal Mf

(A)Macrophages from peritoneal extrudate of Balb/c mouse were infected with parasites. Cultures were treated with DIM, DPDIM, DMDIM and DMODIM separately as indicated in Materials and Methods. Incubations were carried out for 24 hours. Cells were fixed, stained with Giemsa and counted under bright field microscope. Dose dependent amastigote clearance by DIM and its derivatives from infected macrophages were plotted.

(B) Growth of DIM resistant L. donovani axenic amastigotes were monitored inpresence of increasing concentrations (2, 5, 10, 20 and 50 mM) of DIM, DPDIM, DMDIM and DMODIM for 12 h.

PLoS One. 2011;6(12):e28493

DIM derivatives-induced fragmentation of genomic DNA in wild type and DIM resistant parasites.

Relative percentage of DNA fragmentation measured by cell death detection ELISA kit in L. donovani promastigotes treated with DIM, DPDIM, DMDIM and DMODIM.

The experiments were performed three times.

Conclusion

DIM is an antileishmanial agent and is a Class I inhibitor of topoisomerase IB.

DIM inhibits single turnover cleavage and religation activity.

Continuous adaptation with DIM generates DIM-resistant mutant promastigotes (LdDR50).

DIM accumulation remains same in WT and resistant parasites.

ABC Transporters are not involved in the resistance of parasites to DIM.

Topoisomerase I gene mutations at F270 in the large subunit and N184 in the small subunit contribute to resistance towards DIM.

Several derivatives of DIM synthesized, three derivatives identified (DPDIM, DMDIM and DMODIM) which kills resistant parasites, DPDIM being the most potent.

Docking studies provide a possible explanation for higher efficacy of DPDIM.

1. Indolyl Quinolines( synthetic)Dual inhibitor of Topoisomerases, Class II type (BBRC, 1997)

2. Diospyrin- A bis-Naphthoquinone(Diospyros montana)Topoisomerase I inhibitor, Class I type (Mol. Pharmacol., 1998)

3. Luteolin- A flavonoid compound (Vitex negundo)Dual inhibitor of topoisomerases, Class I type (Mol. Med., 2000)

4. Betulin and Dihydrobetulinic acid-A pentacyclic triterpenoid(Bacopa monniera) Dual inhibitor of topoisomerases, Class II type (Mol. Med, 2003; Mol Pharmacol, 2010)

ANTILEISHMANIAL COMPOUNDS

5. Diindolylmethane (DIM) Topoisomerase I inhibitor, Class I type (Biochem J., 2008; Mol. Pharmacol, 2009)

6. Niranthin (Phyllanthus amarus)Topoisomerase I inhibitor, Class I type (EMBO Mol Med, 2012)