Molecular Identification of Diethylcarbamazine Drug Target Proteins

Abstract

The purpose of this study was to identify the target(s) of the drug diethylcarbamazine (DEC); a drug used for over 50 years to treat Brugia malayi larval (L3 stage) parasites. The parasites cause a debilitating disease called lymphatic filariasis (LF); the mechanism of drug action is unknown. The World Health Organization is currently administering a program whereby DEC is dispensed on a wide scale. This may foster the development of drug resistant strains of B. malayi, which causes a major problem. No other drug effectively kills B. malayi. This study was based upon two hypotheses: 1) The drug target of DEC is a protein(s) that can be identified by screening the phage cDNA library of L3 and 2) identification of the drug target(s) of DEC will aid in new drug design and may be potential vaccine candidate(s). Phage display cDNA expression library was screened using biotinylated DEC to identify the proteins. BmMAK-16 and BmSPARC were found to be two drug targets of DEC. Phylogenetic and taxonomic analyses proved that MAK-16 and SPARC mouse and human proteins are distant from B. malayi on the evolutionary tree. Thus, the DEC drug targets may not cause cross-reactivity in humans; making a vaccine more feasible. The function of BmMAK-16 is uncertain. Bioinformatics analyses and literature reviews indicate that MAK-16 and SPARC protein classes have important metabolic functions, making them credible targets for drug and vaccine development. Immunization with rBmMAK-16 and rBmMAK-16 + rBmSPARC developed significant antibody titers of IgG1 and IgG2a in mice. In vitro antibody-dependent cellular cytotoxicity analyses against L3 showed cytotoxicity with rBmMAK-16 (53%) and BmMAK-16 + rBmSPARC (37%). In situ micropore chamber analysis confirmed rBmMAK-16 immunized mice (70%) had greater protection compared to rBmMAK-16 + rBmSPARC (32%). Spleen cells from rBmMAK-16 vaccinated mice showed increased proliferation when stimulated with rBmMAK-16, indicating that memory cells are present. Spleen cells from rBmMAK-16 + rBmSPARC vaccinated mice stimulated with rBmMAK-16 expressed and secreted high levels of IFN-γ suggesting that a Th1 response is predominant. These results show that rBmMAK-16 may be an important target for designing new drugs or vaccines against LF.