Inhibiting parasite proliferation using a rationally designed anti‐tubulin agent

Abstract

Infectious diseases caused by apicomplexan parasites remain a global public health threat. The presence of multiple ligand‐binding sites in tubulin makes this protein an attractive target for anti‐parasite drug discovery. However, despite remarkable successes as anti‐cancer agents, the rational development of protozoan parasite‐specific tubulin drugs has been hindered by a lack of structural and biochemical information on protozoan tubulins. Here, we present atomic structures for a protozoan tubulin and microtubule and delineate the architectures of apicomplexan tubulin drug‐binding sites. Based on this information, we rationally designed the parasite‐specific tubulin inhibitor parabulin and show that it inhibits growth of parasites while displaying no effects on human cells. Our work presents for the first time the rational design of a species‐specific tubulin drug providing a framework to exploit structural differences between human and protozoa tubulin variants enabling the development of much‐needed, novel parasite inhibitors.

Systematic Analysis of Clemastine, a Candidate Apicomplexan Parasite-Selective Tubulin-Targeting Agent

“…We enumerated the number of GFP-expressing parasites in individual vacuoles at 12-hour intervals over one complete lytic cycle. After tracking replication of the parasites treated with clemastine or astemizole for up to 36 h, we did not detect overt slowing of replication, such as was observed for parabulin [ 15 ], nor did we observe aberrant parasite morphology, which is seen soon after oryzalin treatment ( Supplemental Figure S3B ) [ 16 ].…”

“…Subpellicular microtubules from vehicle-treated control cultures have an average length of ~4 μm ( Figure 4 B). Oryzalin-treated parasites exhibit a dose-dependent shortening of microtubule length, and we detected a significant reduction in microtubule length in tachyzoites grown in parabulin, a newly identified compound that selectively targets the protozoan tubulin colchicine site [ 15 ]. Tachyzoites grown in clemastine or astemizole exhibited smaller but significant decreases in filament length in comparison to untreated samples.…”

“…We and others previously described the selective disruption of protozoan-parasite microtubules by oryzalin and other dinitroanilines [ 8 , 12 , 13 , 14 , 36 , 37 ]. More recently, parabulin, a parasite-selective TTA, was shown to inhibit Toxoplasma tachyzoite replication [ 15 ]. As proof of concept for the testing cascade, we investigated whether clemastine indirectly targets Toxoplasma tubulin by impairing dimer folding and, in turn, limiting microtubule assembly.…”

“…This assay detects a dose-dependent shortening of subpellicular microtubules from tachyzoites grown in oryzalin, a well-established direct microtubule-disrupting agent ( Figure 3 B). We recently characterized parasite growth inhibition induced by parabulin, a rationally designed parasite-selective colchicine-site ligand [ 15 ]. Although we could not discern a difference in the morphology of intact tachyzoites after growth in parabulin, we detected significant microtubule shortening relative to control samples.…”

“…However, their ability to distinguish vertebrate from protozoan tubulin demonstrates that identification and use of compounds that selectively affect protozoan parasite microtubules is practical. More recently, parabulin, a compound designed to distinguish the contours of the protozoan colchicine pocket over the vertebrate binding site, was shown to selectively target protozoan tubulin and inhibit the Toxoplasma lytic cycle [ 15 ].…”