Naveen Kumar Kaushik

Synthesis of novel α-pyranochalcones and pyrazoline derivatives as Plasmodium falciparum growth inhibitors

Both the lack of a credible malaria vaccine and the emergence and spread of parasites resistant to most of the clinically used antimalarial drugs and drug combination have aroused an imperative need to develop new drugs against malaria. In present work, alpha-pyranochalcones and pyrazoline analogs were synthesized to discover chemically diverse antimalarial leads. Compounds were tested for antimalarial activity by evaluation of the growth of malaria parasite in culture using the microtiter plate based SYBR-Green-I assay. The (E)-3-(3-(2,3,4-trimethoxyphenyl)-acryloyl)-2H-chromen-2-one (Ga6) turned out to be the most potent analog of the series, showing IC(50) of 3.1 microg/ml against chloroquine-sensitive (3D7) strain and IC(50) of 1.1 microg/ml against chloroquine-resistant field isolate (RKL9) of Plasmodium falciparum. Cytotoxicity study of the most potent compounds was also performed against HeLa cell line using the MTT assay. All the tested compounds showed high therapeutic indices suggesting that they were selective in their action against the malaria parasite. Furthermore, docking of Ga6 into active site of falcipain enzyme revealed its predicted interactions with active site residues. This is the first instance wherein chromeno-pyrazolines have been found to be active antimalarial agents. Further exploration and optimization of this new lead could provide novel, antimalarial molecules which can ward off issues of cross-resistance to drugs like chloroquine.

Reinvestigation of structure-activity relationship of methoxylated chalcones as antimalarials: synthesis and evaluation of 2,4,5-trimethoxy substituted patterns as lead candidates derived from abundantly available natural β-asarone.

We have examined the antimalarial structure-activity relationship of a series of methoxylated chalcones (A-CHCH-CO-B) against Plasmodium falciparum (3D7 strain) using fluorescence-based SYBR Green assay. Our study has revealed that electron releasing methoxy groups on ring A and electron withdrawing groups on ring B increases antimalarial potency while the positional interchange of these groups causes a decrease. In particular, 2,4,5-trimethoxy substitution pattern at ring A provided potent analogues which were easily derived from abundantly available natural β-asarone rich Acorus calamus oil. Cytotoxic evaluation indicated that the most active compounds 27 (IC(50): 1.8 μM) and 26 (IC(50): 2 μM) were also relatively non-toxic. Furthermore, compound 12 showed excellent resistance index of 1.1 against chloroquine resistant Dd2 strain of P. falciparum.

Evaluation of antiplasmodial activity of green synthesized silver nanoparticles.

In the present study silver nanoparticles (silver(np)) were synthesized from AgNO3 through simple green routes using either purified Alpha Amylase or aqueous leaf extracts of Ashoka and Neem respectively. The use of plant extract/enzyme for synthesis of nanoparticles is a single-step, cost effective and eco-friendly process. The silver(np) obtained by these three different ways were characterized using UV-visible spectroscopy, DLS, TEM, XRD and FTIR. These nanoparticles were found to be antiplasmodial with IC50 (μg/ml) 3.75 (Amylase(np)), 8 (Ashoka(np)) and 30 (Neem(np)) whereas plant extracts or amylase alone did not show any activity up to 40 μg/ml. Although AgNO3 was also found to have intrinsic antiplasmodial activity (IC50 0.5 μg/ml), the hemolytic tendencies appeared to be higher for AgNO3 (MHC10: 10 μg/ml) against the nanoparticulate preparations (MHC10: >40 μg/ml).

Antimalarial activities of medicinal plants traditionally used in the villages of Dharmapuri regions of South India.

ETHNOPHARMACOLOGICAL RELEVANCE An ethnopharmacological investigation of medicinal plants traditionally used to treat diseases associated with fevers in Dharmapuri region of South India was undertaken. Twenty four plants were identified and evaluated for their in vitro activity against Plasmodium falciparum and assessed for cytotoxicity against HeLa cell line. AIM OF THE STUDY This antimalarial in vitro study was planned to correlate and validate the traditional usage of medicinal plants against malaria. MATERIALS AND METHODS An ethnobotanical survey was made in Dharmapuri region, Tamil Nadu, India to identify plants used in traditional medicine against fevers. Selected plants were extracted with ethyl acetate and methanol and evaluated for antimalarial activity against erythrocytic stages of chloroquine (CQ)-sensitive 3D7 and CQ-resistant INDO strains of Plasmodium falciparum in culture using the fluorescence-based SYBR Green I assay. Cytotoxicity was determined against HeLa cells using MTT assay. RESULTS Promising antiplasmodial activity was found in Aegle marmelos [leaf methanol extract (ME) (IC(50)=7 μg/mL] and good activities were found in Lantana camara [leaf ethyl acetate extract (EAE) IC(50)=19 μg/mL], Leucas aspera (flower EAE IC(50)=12.5 μg/mL), Momordica charantia (leaf EAE IC(50)=17.5 μg/mL), Phyllanthus amarus (leaf ME IC(50)=15 μg/mL) and Piper nigrum (seed EAE IC(50)=12.5 μg/mL). The leaf ME of Aegle marmelos which showed the highest activity against Plasmodium falciparum elicited low cytotoxicity (therapeutic index>13). CONCLUSION These results provide validation for the traditional usage of some medicinal plants against malaria in Dharmapuri region, Tamil Nadu, India.

Pyrano(4,3-b)quinolines Library Generation via Iodocyclization and Palladium-Catalyzed Coupling Reactions

Synthesis of a 80-member library of novel pyrano[4,3-b]quinolines in solution-phase is reported. The key intermediate, 4-iodopyrano[4,3-b]quinolines were synthesized by the electrophilic iodocyclization of corresponding ortho-alkynyl aldehydes in good to excellent yields under mild reaction conditions. Subsequently a diverse set of libraries was generated by employing palladium-catalyzed Suzuki-Miyaura, Heck, and Sonogashira coupling reactions on 4-iodopyrano[4,3-b]quinolines. In this way, a series of structurally different and biologically interesting molecules were obtained. Some of the selected compounds were screened against 3D7 strains of Plasmodium falciparum for antimalarial activity. Suzuki coupling products 6{3} and 6{21} and Heck coupling product 8{12} exhibit promising antimalarial activity.

Plasmodium falciparum merozoite surface protein 3: oligomerization, self-assembly, and heme complex formation.

Background: Plasmodium falciparum merozoite surface protein 3 (MSP3) oligomerizes and binds heme. Results: MSP3 forms amyloid-like structures that bind ∼35 heme molecules, and these filamentous structures are also present on the surface of merozoites. Conclusion: Amyloid formation by MSP3 is related to heme binding. Significance: The presence of MSP3 fibrils on merozoite surface and significant heme binding suggest its functional role during erythrocytic stages of P. falciparum. Merozoite surface protein 3 of Plasmodium falciparum, a 40-kDa protein that also binds heme, has been biophysically characterized for its tendency to form highly elongated oligomers. This study aims to systematically analyze the regions in MSP3 sequence involved in oligomerization and correlate its aggregation tendency with its high affinity for binding with heme. Through size exclusion chromatography, dynamic light scattering, and transmission electron microscopy, we have found that MSP3, previously known to form elongated oligomers, actually forms self-assembled filamentous structures that possess amyloid-like characteristics. By expressing different regions of MSP3, we observed that the previously described leucine zipper region at the C terminus of MSP3 may not be the only structural element responsible for oligomerization and that other peptide segments like MSP3(192–196) (YILGW) may also be required. MSP3 aggregates on incubation were transformed to long unbranched amyloid fibrils. Using immunostaining methods, we found that 5–15-μm-long fibrillar structures stained by anti-MSP3 antibodies were attached to the merozoite surface and also associated with erythrocyte membrane. We also found MSP3 to bind several molecules of heme by UV spectrophotometry, HPLC, and electrophoresis. This study suggested that its ability to bind heme is somehow related to its inherent characteristics to form oligomers. Moreover, heme interaction with a surface protein like MSP3, which does not participate in hemozoin formation, may suggest a protective role against the heme released from unprocessed hemoglobin released after schizont egress. These studies point to the other roles that MSP3 may play during the blood stages of the parasite, in addition to be an important vaccine candidate.

Dibenzylideneacetone analogues as novel Plasmodium falciparum inhibitors

A series of dibenzylideneacetones (A1-A12) and some of their pyrazolines (B1-B4) were synthesized and evaluated in vitro for blood stage antiplasmodial properties in Plasmodium falciparum culture using SYBR-green-I fluorescence assay. The compound (1E, 4E)-1,5-bis(3,4-dimethoxyphenyl)penta-1,4-dien-3-one (A9) was found to be the most active with IC(50) of 1.97 μM against chloroquine-sensitive strain (3D7) and 1.69 μM against chloroquine-resistant field isolate (RKL9). The MTT based cytotoxicity assay on HeLa cell line has confirmed that A9 is selective in its action against malaria parasite (with a therapeutic index of 166). Our results revealed that these compounds exhibited promising antiplasmodial activities which can be further explored as potential leads for the development of cheaper, safe, effective and potent drugs against chloroquine-resistant malarial parasites.

A search for antiplasmodial metabolites among fungal endophytes of terrestrial and marine plants of southern India.

Eighty four different fungal endophytes isolated from sea grasses (5), marine algae (36) and leaves or barks of forest trees (43) were grown in vitro and the secondary metabolites secreted by them were harvested by immobilizing them on XAD beads. These metabolites were eluted with methanol and screened using SYBR Green I assay for their antiplasmodial activity against blood stage Plasmodium falciparum in human red blood cell culture. Our results revealed that fungal endophytes belonging to diverse genera elaborate antiplasmodial metabolites. A Fusarium sp. (580, IC50: 1.94 μg ml−1) endophytic in a marine alga and a Nigrospora sp. (151, IC50: 2.88 μg ml−1) endophytic in a tree species were subjected to antiplasmodial activity-guided reversed phase high performance liquid chromatography separation. Purification led to potentiation as reflected in IC50 values of 0.12 μg ml-1 and 0.15 μg ml−1 for two of the fractions obtained from 580. Our study adds further credence to the notion that fungal endophytes are a potential storehouse for a variety of novel secondary metabolites vested with different bioactivities including some that can stall the growth of the malaria parasite.

Antiplasmodial potential of selected medicinal plants from Eastern Ghats of South India

Malaria caused by the protozoan parasite Plasmodium falciparum, is a major health problem of the developing world. In the present study medicinal plants from Eastern Ghats of South India have been extracted with ethyl acetate and assayed for growth inhibition of asexual erythrocytic stages of chloroquine (CQ)-sensitive (3D7) and (CQ)-resistant (INDO) strains of P. falciparum in culture using the fluorescence-based SYBR Green I assay. Studied extracts showed a spectrum of antiplasmodial activities ranging from (a) very good (IC(50)<10-10 μg/mL: Cyperus rotundus and Zingiber officinale); (b) good (IC(50), >10-15 μg/mL: Ficus religiosa and Murraya koenigii); (c) moderate (IC(50)>15-25 μg/mL: Ficus benghalensis); (d) poor activity (IC(50)>25-60 μg/mL) and (e) inactive (IC(50)>60 μg/mL). Resistance indices ranging from 0.78 to 1.28 suggest that some of these extracts had equal promise against the CQ resistant INDO strain of P. falciparum. Cytotoxicity assessment of the extracts against HeLa cell line using MTT assay revealed that the selectivity indices in the range of 3-15 suggesting a good margin of safety.

Antiplasmodial activity of medicinal plants from Chhotanagpur plateau, Jharkhand, India.

BACKGROUND The alarmingly increasing problem of drug resistance in treatment of malaria has led to an urgent need for identifying new anti-malarial drugs for both prophylaxis and chemotherapy. AIM OF THE STUDY The present study presents a systematic exploration of the ex vivo blood stage antiplasmodial potential of medicinal plants to corroborate their traditional usage against malaria in Jharkhand, India. METHODS An ethnobotanical survey in and around Ranchi was done to grasp the traditional knowledge of medicinal plants used by local healers for malaria, other fevers and for other medicinal purposes like, antiamoebic, antihelmenthic, antidote to poisons, etc. Following the survey, the selected 22 plant samples were extracted in ethanol for studying ex vivo SYBR Green I fluorescence assay based anti-plasmodial activity against both chloroquine-sensitive Pf3D7 and chloroquine resistant PfINDO strains of Plasmodium falciparum grown in human red blood cell cultures. Cytotoxicity was determined against HeLa and L929 cells using MTT assay. Further the most potent extract was chromatographed on reverse phase HPLC towards antiplasmodial activity guided purification of metabolites. RESULTS Of the 22 plant species assayed, the highest antiplasmodial activity (Pf3D7IC50 ≤ 5 µg/ml) was seen in leaf ethanol extracts of Corymbia citriodora (Hook.) K.D.Hill & L.A.S.Johnson, Calotropis procera (Aiton) Dryand. and Annona squamosa L. and bark ethanol extract of Holarrhena pubescens Wall. ex G.Don. Leaf ethanol extract of H. pubescens, bark ethanol extract of Pongamia pinnata (L.) Pierre and whole plant ethanol extract of Partheniumhysterophorus L. showed promising activity (IC50 6-10 µg/ml). Good antiplasmodial activity (IC50: 11-20 µg/ml) was observed in leaf ethanol extract of Bryophyllum pinnatum (Lam.) Oken and whole plant ethanol extract of Catharanthus roseus (L.) G.Don. The extracts of plants showing highest to good antiplasmodial activity exhibited HeLa/Pf3D7 selectivity indices of the order of 20-45. Bioassay guided fractionation of P. hysterophorus led to fivefold enrichment of antiplasmodial activities (IC50 ~450 ng/ml) in some fractions. CONCLUSION These results provide confirmation to the traditional usage of some medicinal plants against malaria in areas around Ranchi, Jharkhand.