Abstract
Plocabulin (PM060184) is a microtubule depolymerizing agent with potent antiproliferative activity undergoing phase II clinical trials for the treatment of solid tumors. Plocabulin shows antifungal activity virtually abolishing growth of the filamentous fungus Aspergillus nidulans. A. nidulans hyphae depend both on mitotic and interphase microtubules, as human cells. Here, we exploited the A. nidulans genetic amenability to gain insight into the mechanism of action of plocabulin. By combining mutations in the two A. nidulans β-tubulin isotypes we obtained a plocabulin-insensitive strain, showing that β-tubulin is the only molecular target of plocabulin in fungal cells. From a genetic screen, we recovered five mutants that show plocabulin resistance but do not carry mutations in β-tubulin. Resistance mutations resulted in amino acid substitutions in (1) two subunits of the eukaryotic translation initiation factor eIF2B activating the General Amino Acid Control, (2) TIM44, an essential component of the inner mitochondrial membrane translocase, (3) two transcription factors of the binuclear zinc cluster family potentially interfering with the uptake or efflux of plocabulin. Given the conservation of some of the identified proteins and their respective cellular functions in the tumor environment, our results pinpoint candidates to be tested as potential biomarkers for determination of drug efficiency.
Highlights
Plocabulin (PM060184, PM184) (Fig. 1a) is a microtubule (MT) depolymerizing agent currently undergoing phase II clinical trials for the treatment of solid tumors
As a proof of concept we had previously isolated from a random genetic screen plocabulin-resistant A. nidulans mutants that constituted in vivo evidence for the existence of a novel plocabulin specific binding site on β-tubulin
The crystal structures of tubulin bound to plocabulin, rhizoxin and maytansin were consistent with these conclusions, showing that Asn[100] is part of the β-tubulin binding site that contacts a common pharmacophore on these compounds[2]
Summary
Plocabulin (PM060184, PM184) (Fig. 1a) is a microtubule (MT) depolymerizing agent currently undergoing phase II clinical trials for the treatment of solid tumors. As a proof of concept we had previously isolated from a random genetic screen plocabulin-resistant A. nidulans mutants that constituted in vivo evidence for the existence of a novel plocabulin specific binding site on β-tubulin. These strains carried mutations leading to the Asn100Ile substitution in the major β-tubulin BenA [reported in]3, a substitution shown to confer resistance to rhizoxin[17]. Asn[100] in BenA is critical for plocabulin binding, A. nidulans carrying benAN100I is only partially resistant to the drug[3] (Fig. 1b), suggesting that intracellular targets other than BenA would contribute to plocabulin antifungal activity. We establish a causal relationship between plocabulin partial resistance and mutations in (1) two subunits of the eukaryotic translation initiation factor eIF2B, (2) TIM44, a conserved and essential component of the translocase of the inner mitochondrial membrane, and (3) two DNA binding domain-containing proteins of the zinc binuclear cluster family
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