title: Development of Leishmania donovani stably expressing DsRed for flow cytometry-based drug screening using chalcone thiazolyl-hydrazone as a new antileishmanial target.
abstract: Green fluorescent protein produces significant fluorescence and is extremely stable, however its excitation maximum is close to the ultraviolet range and thus can damage living cells. Hence, Leishmania donovani stably expressing DsRed were developed and their suitability for flow cytometry-based antileishmanial screening was assessed by evaluating the efficacies of standard drugs as well as newly synthesised chalcone thiazolyl-hydrazone compounds. The DsRed gene was successfully integrated at the 18S rRNA locus of L. donovani and transfectants (LdDsRed) were selected using hygromycin B. Enhanced expression of DsRed and a high level of infectivity to J774A.1 macrophages were achieved, which was confirmed by fluorescence microscopy and flow cytometry. Furthermore, these LdDsRed transfectants were utilised for development of an in vitro screening assay using the standard antileishmanial drugs miltefosine, amphotericin B, pentamidine and paromomycin. The response of transfectants to standard drugs correlated well with previous reports. Subsequently, the suitability of this system was further assessed by screening a series of 18 newly synthesised chalcone thiazolyl-hydrazone compounds in vitro for their antileishmanial activity, wherein 8 compounds showed moderate antileishmanial activity. The most active compound 5g, with ca. 73% splenic parasite reduction, exerted its activity via generating nitric oxide and reactive oxygen species and inducing apoptosis in LdDsRed-infected macrophages. Thus, these observations established the applicability of LdDsRed transfectants for flow cytometry-based antileishmanial screening. Further efforts aimed at establishing a high-throughput screening assay and determining the in vivo screening of potential antileishmanial leads are required.
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India.
authors.forename: Anil Kumar
authors.initials: AK
authors.lastname: Jaiswal
authors.affiliation: Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow, India.
authors.forename: K Bhaskara
authors.initials: KB
authors.lastname: Rao
authors.affiliation: Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow, India.
authors.forename: Pragati
authors.initials: P
authors.lastname: Kushwaha
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India.
authors.forename: Keerti
authors.initials: K
authors.lastname: Rawat
authors.affiliation: Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow, India.
authors.forename: Ram K
authors.initials: RK
authors.lastname: Modukuri
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India.
authors.forename: Prashant
authors.initials: P
authors.lastname: Khare
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India.
authors.forename: Sumit
authors.initials: S
authors.lastname: Joshi
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India.
authors.forename: Shikha
authors.initials: S
authors.lastname: Mishra
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India.
authors.forename: Ambak
authors.initials: A
authors.lastname: Rai
authors.affiliation: Division of Medicinal and Process Chemistry, Central Drug Research Institute, Lucknow, India. Electronic address: kv_sashidhara@cdri.res.in.
authors.forename: Koneni V
authors.initials: KV
authors.lastname: Sashidhara
authors.affiliation: Division of Parasitology, Central Drug Research Institute, Lucknow, India. Electronic address: anuradha_dube@hotmail.com.
authors.forename: Anuradha
authors.initials: A
authors.lastname: Dube
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000008:administration & dosage / Q000302:isolation & purification / Q000494:pharmacology*; D002460:Cell Line; D002599:Chalcone / Q000008:administration & dosage / Q000494:pharmacology*; D006224:Cricetinae; D016054:DNA, Protozoan / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004195:Disease Models, Animal; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D017930:Genes, Reporter; D006835:Hydrazones / Q000008:administration & dosage / Q000494:pharmacology; D007893:Leishmania donovani / Q000187:drug effects* / Q000235:genetics; D007898:Leishmaniasis, Visceral / Q000188:drug therapy / Q000469:parasitology; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics; D008264:Macrophages / Q000469:parasitology; D008297:Male; D051379:Mice; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D011995:Recombination, Genetic; D013194:Staining and Labeling / Q000379:methods*; D016896:Treatment Outcome
chemical_list: D000981:Antiprotozoal Agents; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D006835:Hydrazones; D008164:Luminescent Proteins; D012337:RNA, Ribosomal, 18S; C413663:fluorescent protein 583; D002599:Chalcone
keywords: Chalcone thiazolyl-hydrazone; DsRed-Express-N1; Leishmania donovani; Mean fluorescence intensity; Multiparameter flow cytometry; Red fluorescent protein