New Class of Antitrypanosomal Agents Based on Imidazopyridines

The present work describes the synthesis of 22 new imidazopyridine analogues arising from medicinal chemistry optimization at different sites on the molecule. Seven and 12 compounds exhibited an in vitro EC₅₀ ≤ 1 μM against <i>Trypanosoma cruzi</i> (<i>T. cruzi</i>) and <i>Trypanosoma brucei</i> (<i>T. brucei</i>) parasites, respectively. Based on promising results of <i>in vitro</i> activity (EC₅₀ < 100 nM), cytotoxicity, metabolic stability, protein binding, and pharmacokinetics (PK) properties, compound <b>20</b> was selected as a candidate for <i>in vivo</i> efficacy studies. This compound was screened in an acute mouse model against <i>T.cruzi</i> (<i>Tulahuen</i> strain). After established infection, mice were dosed twice a day for 5 days, and then monitored for 6 weeks using an <i>in vivo</i> imaging system (IVIS). Compound <b>20</b> demonstrated parasite inhibition comparable to the benznidazole treatment group. Compound <b>20</b> represents a potential lead for the development of drugs to treat trypanosomiasis

Analysis of Rab prenylation rates <i>in</i><i>vivo</i> and <i>in</i><i>vitro</i>.

<p>(A-B) Analysis of Rab prenylation status after blocking and releasing prenylation <i>in </i><i>vivo</i>. HeLa cells were treated with compactin for 24h and then incubated with GGOH for different time periods. Cells were lysed and subjected to <i>in </i><i>vitro</i> prenylation with BGPP and recombinant RabGGTase and REP for 6h. (A) Degree of prenylation for each Rab was determined by mass spectrometry. The decrease in signal from the timepoint 0h to 5h was determined by label-free spectral counting and converted into the degree of prenylation for each Rab 5 hours after GGOH addition. The graph represents the mean of three independent experiments (±SEM). (B) Streptavidin-HRP Western blot detection of unprenylated Rabs in the cellular lysates at different timepoints after GGOH addition to compactin-treated cells. The cellular lysates were prenylated with BGPP and RabGGTase as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081758#pone-0081758-g003" target="_blank">Figure 3 (C)</a><i>In </i><i>vitro</i> prenylation of lysate from compactin-treated HeLa cells transfected with different GFP-Rabs. <i>In </i><i>vitro</i> prenylation reaction was stopped after an hour and subjected to Western blot analysis visualized by infrared Odyssey scanning for total GFP-Rab (GFP/red) and for prenylated biotin-labeled GFP-Rab (biotin/green). Representative blots are shown. (D) The graph represents the percentage of prenylated GFP-Rabs after an hour <i>in </i><i>vitro</i> prenylation reaction normalized to complete (overnight) prenylation. Means of three independent experiments are shown (±SEM).</p

Rescue of underprenylation in REP1 knockdown cells by REP1 but not by REP2.

<p>(A) REP1 knockdown cells were transfected with rat REP1-myc, REP2-myc or the vector control. The cell lysate was subjected to the <i>in </i><i>vitro</i> prenylation with BGPP to quantitate the levels of unprenylated Rabs. (B) Quantification of unprenylated Rabs in REP1 knockdown cells transfected with REP1 or REP2. The Western Blot signal of the unprenylated Rabs was normalized to the β-actin signal to account for differences in loading. REP1 knockdown cells transfected with vector only were taken as reference representing maximal underprenylation of Rabs in this model and therefore shown as 100%.The graph represents the mean of three independent experiments (± SEM). P-levels are denoted above the bars and were determined with the two-tailed Student’s t-test.</p

Schematic representation of <i>in</i><i>vitro</i> prenylation assay.

<p>(A) Cell lysates are subjected to <i>in </i><i>vitro</i> prenylation using a recombinant Rab prenylation machinery and the biotin-labeled analogue of GGPP, BGPP. Only those Rabs unprenylated in the cell will be labeled in the assay and can be visualized or purified through the biotin-tag. (B) Western blot analysis of cell lysate from untreated and compactin-treated BHK cells after <i>in </i><i>vitro</i> prenylation with BGPP. Unprenylated Rabs are detected via the biotin-tag and actin serves as a loading control. BGPP: biotin-geranyl-pyrophosphate, GGPP: geranylgeranyl-pyrophosphate. Note that compactin treated sample is underloaded and the actual level of unprenylated RabGTPases are higher than they appear from comparison of the band’s intensities. </p

Localization is an indicator of prenylation.

<p>MALDI mass spectrometry comparison of unmodified (gray) and <i>in </i><i>vitro</i> prenylated (black) Citrine-Rab7 (A) and Citrine-Rab27a (B). Average protein masses are indicated at the peaks. Prenylated Citrine-Rab7:REP1 (C) and prenylated Citrine-Rab27a:REP1 (D) were microinjected into compactin treated A431 cells. Membrane targeting of Rab proteins was detected by imaging the cells 20 min post-injection. Scale bars represent 10 µm.</p

Rates of <i>in</i><i>vivo</i> prenylation and localization for Rab1a, Rab7a and Rab27a.

<p>Unprenylated, fluorescently-tagged recombinant CFP-Rab1a (upper panel), YFP-Rab7a (middle panel) and Cherry-Rab27a (lower panel) were microinjected into A431 cells. Cells were imaged over 22h and cellular localization of the injected Rab proteins detected by their fluorescent tags. The timepoints after microinjection are displayed above the images in hours or overnight (ON). Cells were pre-incubated with compactin where stated. Scale bars represent 20 µm.</p