Selective Antimicrotubule Activity ofN1-Phenyl-3,5-dinitro-N4,N4-di-n-propylsulfanilamide (GB-II-5) against Kinetoplastid Parasites

Abstract

Analogs of the antimitotic herbicide oryzalin (3,5-dinitro-N4,N4-di-n-propylsulfanilamide) were recently prepared that were more potent in vitro than the parent compound against the kinetoplastid parasite Leishmania donovani (Bioorg Med Chem Lett 12:2395-2398. In the present work, we show that the most active molecule in the group,

Leishmania tarentolae: Purification and characterization of tubulin and its suitability for antileishmanial drug screening

“…A comparison of these sequences provides some insight into similarities and differences in antimitotic drug susceptibility among these organisms. Compounds that are effective antimitotic agents against Leishmania would also be expected to be effective against African trypanosomes, as has been observed for GB-II-5 and GB-II-150 (Werbovetz et al, 2003;Bhattacharya et al, 2004) based on the 94% amino acid identity between the α-and β-tubulin sequences from T. brucei and L. tarentolae. The lower amino acid sequence identity between L. tarentolae and mammalian tubulin (81% and 84% for α-and β-tubulin, respectively) is reflected by differences in susceptibility to antimitotic ligands.…”

“…Emission intensity at 325 nm was taken for calculation of quenching. In contrast to the previous study (Werbovetz et al, 2003), multiple tubulin samples with varying concentrations of compound in a fixed volume of solvent (DMSO, 9.7% v/v) were prepared. Since the DMSO concentrations were fixed and the fluorescence was only measured once per sample, it was unnecessary to correct for solvent effects and photobleaching.…”

“…Quenching of intrinsic tubulin fluorescence by dinitroaniline compounds was assessed based on a previous method (Werbovetz et al, 2003). Samples were studied using sub-micro 50 μL quartz spectrophotometer cells (Starna Cells).…”

“…Tubulin/drug assembly assays were performed as outlined previously (Werbovetz et al, 2003). Assembly reactions were carried out in 96-well half-area microplates (Costar) in a final volume of 50 μL and contained final concentrations of 1.5 mg/ml (15 μM) tubulin, 0.1 mM PIPES (pH 6.9), 1 mM EGTA, 1 mM MgCl 2 , 10% (v/v) DMSO, and 1 mM GTP with and without compounds of interest.…”

“…Dinitroaniline herbicides have shown activity versus both Leishmania and trypanosomes (Chan and Fong, 1990;Chan et al, 1991;Chan et al, 1993a;Chan et al, 1993b;Traub-Cseko et al, 2001), and tubulin has been implicated as the target of these compounds (Chan and Fong, 1990;Chan et al, 1991;Traub-Cseko et al, 2001). N 1 -phenyl-3,5-dinitro-N 4 ,N 4 -di-n-propylsulfanilamide (GB-II-5) (Werbovetz et al, 2003) and N 1 -phenyl-3,5-dinitro-N 4 ,N 4 -di-n-butylsulfanilamide (GB-II-150) (Bhattacharya et al, 2004) are derivatives of the dinitroaniline herbicide oryzalin that have displayed potent antimitotic effects on kinetoplastid parasites (see Figure 1 for structures). Initially, these oryzalin analogs were evaluated in vitro for their ability to inhibit the polymerization of tubulin purified from L. amazonensis (Werbovetz et al, 1999), and ongoing drug discovery efforts against this promising target will continue to require in vitro evaluation of novel compounds against purified leishmanial tubulin.…”